Plant with altered content of steroidal alkaloids

ABSTRACT

The present invention relates to key genes in the biosynthesis of steroidal alkaloids and saponins, including regulatory genes and enzyme-encoding genes, and to use thereof for altering the content of steroidal (glyco)alkaloids or phytosterols in plants. The present invention provides genetically modified plants or gene edited plants with altered content of steroidal (glyco)alkaloids, particularly to Solanaceous crop plants with reduced content of antinutritional steroidal glycoalkaloids and to the increase in phytosterols, including cholesterol or cholestanol in these plants. The present invention also provides methods of altering gene expression.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 14/895,059 filed Dec. 1, 2015, which filed as aNational Phase Application of PCT International Application NumberPCT/IL2014/050497, International filing date Jun. 2, 2014, which claimsthe benefit of U.S. Provisional Application Ser. No. 61/831,164 filedJun. 5, 2013; which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to key genes in the biosynthesis ofsteroidal alkaloids and steroidal saponins and to genetically modifiedor gene edited plants with altered content of steroidal alkaloids,steroidal saponins, or phytosterols, particularly to Solanaceous cropplants with reduced content of antinutritional steroidal glycoalkaloidsor increased content of phytosterols, including cholesterol,cholestanol, and any of their modified glycosylated derivatives.

BACKGROUND OF THE INVENTION

The plant kingdom produces hundreds of thousands of different smallcompounds that are often genus or family specific. These molecules,referred to as secondary metabolites, are not vital to cells thatproduce them, but contribute to the overall fitness of the organisms.Alkaloids are one example of secondary metabolites. They are lowmolecular weight nitrogen-containing organic compounds, typically with aheterocyclic structure. Alkaloid biosynthesis in plants is tightlycontrolled during development and in response to stress and pathogens.

The broad group of triterpenoid-alkaloid compounds is widespread inplants and derived from the cytosolic mevalonic acid isoprenoidbiosynthetic pathway. Steroidal saponins and Steroidal alkaloids are twolarge classes of triterpenoids produced by plants. Steroidal alkaloids(SAs), occasionally known as “Solanum alkaloids,” are commonconstituents of numerous plants belonging to the Solanaceae family,which includes the genera Solanum and Capsicum, as well as many others.Steroidal alkaloids are also produced by a large number of species inthe Liliaceae family.

Estimated in the order of 1350 species, Solanum is one of the largestgenera of flowering plants, representing about a half of the species inthe Solanaceae. Diverse structural composition and biological activity,as well as occurrence in food plants including tomato (Solanumlycopersicum), potato (Solanum tuberosum) and eggplant (Solanummelongena), made SAs the subject of extensive investigations (Eich E.2008. Solanaceae and Convolvulaceae—secondary metabolites: biosynthesis,chemotaxonomy, biological and economic significance: a handbook. Berlin:Springer).

Consisting of a C-27 cholestane skeleton and a heterocyclic nitrogencomponent, SAs were suggested to be synthesized in the cytosol fromcholesterol. Conversion of cholesterol to the alkamine SA should requireseveral hydroxylation, oxidation and transamination reactions (Eich2008, supra), and in most cases further glycosylation to form steroidalglycoalkaloids (SGAs) (Arnqvist L. et al. 2003. Plant Physiol131:1792-1799). The oligosaccharide moiety components of SGAs directlyconjugate to the hydroxyl group at C-3β of the alkamine steroidalskeleton (aglycone). The oligosaccharide moiety includes D-glucose,D-galactose, L-rhamnose, D-xylose, and L-arabinose, the first twomonosaccharides being the predominant units.

Steroidal glycoalkaloids (SGAs) are nitrogen-containing,cholesterol-derived specialized metabolites produced by numerous membersof the Solanaceae family. Examples of these compounds include α-tomatineand dehydrotomatine in tomato (Solanum lycopersicum), α-chaconine andα-solanine in potato (Solanum tuberosum), and α-solamargine andα-solasonine in eggplant (Solanum melongena). SGAs are also found invarious types of peppers in the genus Capsicum. SGAs contribute to plantresistance to a wide range of pathogens and predators, includingbacteria, fungi, oomycetes, viruses, insects, and larger animals. Someof them chaconine and α-solanine in potato) are considered asanti-nutritional compounds to humans due to their toxic effects. Morethan 100 SGAs have been identified in tomatoes (Itkin et al., 2011,Plant Cell 23:4507-4525), and more than 50 have been identified inpotatoes (Shakya and Navarre, 2008, J. Agric. Food Chem. 56:6949-6958).Eggplant also contains at least one variety of SGA (Friedmann, 2006, J.Agric. Food Chem. 54:8655-8681).

SGA biosynthesis depends on genes encoding UDP-glycosyltransferases(UGTs) that decorate the aglycone with various sugar moieties (McCue K Fet al., 2005, Plant Sci. 168:267-273; Itkin M et al., 2011. Plant Cell23:4507-4525). The tomato GLYCOALKALOID METABOLISM 1 (GAME1)glycosyltransferase, a homolog of the potato SGT1 (McCue et al., 2005,supra), catalyzes galactosylation of the alkamine tomatidine (Itkin etal., 2011, supra). SGA biosynthesis depends both on SGA biosynthesisgenes (e.g., GAME 4, GAME12) and on regulators of SGA biosynthesis(e.g., GAME9) (Itkin et al. 2013. Science 341: 175-179; Cardenas et al.2016. Nat. Commun. 7: 10654).

Steroidal alkaloids play a role in protecting plants against a broadrange of pathogens and are thus referred to as phytoanticipins(antimicrobial compounds). Many SGAs are harmful to a variety oforganisms including mammals and humans. When present in edible plantparts, these harmful SGAs are referred to as antinutritional substances.The SGAs α-solanine and α-chaconine are the principle toxic substancesin potato. These SGAs cause gastrointestinal and neurological disordersand, at high concentrations, may be lethal to humans. Mechanisms oftoxicity include disruption of membranes and inhibition of acetylcholineesterase activity (Roddick J G. 1989. Phytochemistry 28:2631-2634). Forthis reason, total SGA levels exceeding 200 mg per kilogram fresh weightof edible tuber are deemed unsafe for human consumption.

There is an ongoing attempt to elucidate the biosynthesis pathway ofsteroidal alkaloids and to control their production. U.S. Pat. No.5,959,180 discloses DNA sequences from potato which encode the enzymesolanidine UDP-glucose glucosyltransferase (SGT). Further disclosed aremeans and methods for inhibiting the production of SGT and therebyreduce glycoalkaloid levels in Solanaceous plants, for example potato.

Similarly, U.S. Pat. Nos. 7,375,259 and 7,439,419 disclose nucleic acidsequences from potato that encode the enzymes UDP-glucose:solanidineglucosyltransferase (SGT2) and β-solanine/β-chaconinerhamnosyltransferase (SGT3), respectively. Recombinant DNA moleculescontaining the sequences, and use thereof, in particular, use of thesequences and antisense constructs to inhibit the production ofSGT2/SGT3 and thereby reduce levels of the predominant steroidalglycoalkaloids α-chaconine and α-solanine in Solanaceous plants such aspotato are also described.

The inventors of the present invention have recently identified threeglycosyltransferases that are putatively involved in the metabolism oftomato steroidal alkaloids (GLYCOALKALOID METABOLISM 1-3 (GAME1-3). Morespecifically, alterations in GAME1 expression modified the SA profile intomato plants in both reproductive and vegetative parts. It is suggestedthat these genes are involved in the metabolism of tomatidine (theα-tomatine precursor) partially by generating the lycotetraose moiety(Itkin et al., 2011, supra).

International Patent Application Publication No. WO 00/66716 discloses amethod for producing transgenic organisms or cells comprising DNAsequences which code for sterol glycosyl-transferases. The transgenicorganisms include bacteria, fungi, plants and animals, which exhibit anincreased production of steroid glycoside, steroid alkaloid and/orsterol glycoside compared to that of wild-type organisms or cells. Thesynthesized compounds are useful in the pharmaceutical and foodstuffindustries as well as for protecting plants.

U.S. Patent Application Publication No. 2012/0159676 discloses a geneencoding a glycoalkaloid biosynthesis enzyme derived from a plantbelonging to the family Solanaceae for example potato (Solanumtuberosum). A method for producing/detecting a novel organism using agene encoding the protein is also disclosed.

U.S. Patent Application Publication No. 2013/0167271 and InternationalApplication Publication No. WO 2012/095843 relate to a key gene in thebiosynthesis of steroidal saponins and steroidal alkaloids and to meansand methods for altering the gene expression and the production ofsteroidal saponins and steroidal alkaloids.

A paper of the inventors of the present invention, published after thepriority date of the present invention, describes an array of 10 genesthat partake in SGA biosynthesis. 5-7 of the genes were found to existas a cluster on chromosome 7 while additional two reside adjacent in aduplicated genomic region on chromosome twelve. Following systematicfunctional analysis, a novel SGA biosynthetic pathway starting fromcholesterol up to the tetrasaccharide moiety linked to the tomato SGAaglycone has been proposed (Itkin M. et al., 2013 Science 341(6142):175-179).

It has also been found that the plant cholesterol biosynthetic pathwayoverlaps with phytosterol metabolism (Sonawane et al. 2016. Nat. Plants3: 16205). For example, cholesterol ((3β)-cholest-5-en-3-ol) is a sterol(or modified steroid), a type of lipid molecule, and is biosynthesizedby all animal cells, because it is an essential structural component ofall animal cell membranes and is essential to maintain both membranestructural integrity and fluidity. It is often found in animal cellmembranes, enabling animal cells to function without a cell wall. It isa precursor for the biosynthesis of steroid hormones, bile acid andvitamin D.

Cholestanol is a cholesterol derivative found in feces, gallstones,eggs, and other biological matter. 5β-Coprostanol (5β-cholestan-3β-ol)is a 27-carbon stanol formed from the biohydrogenation of cholesterol(cholest-5en-3β-ol) in the gut of most higher animals (e.g., birds;humans and other mammals). It is formed by the conversion of cholesterolto coprostanol (cholestanol) in the gut of most higher animals byintestinal bacteria.

Plants make cholesterol in very small amounts, but also manufacturephytosterols (which include plant sterols and stanols, similar tocholesterol and cholestanol), which can compete with cholesterol forreabsorption in the intestinal tract, thus potentially reducingcholesterol reabsorption. Cholesterol is often used in the manufactureof medicines, cosmetics, and other applications. There is an increasedinterest in producing increased levels of both plant phytosterols andplant-based cholesterol.

In tomato (e.g., Solanum lycopersicum, Solanum pennellii), α-tomatineand dehydrotomatine represent the major SGAs accumulating predominantlyin green tissues; young and mature leaves, flower buds, skin and seedsof immature and mature green fruit. Dehydrotomatidine (i.e. tomatidenol)is the first SA aglycone formed in SGA biosynthesis which could furtherbe hydrogenated at the C-5 position to form tomatidine. Both aglyconesare further glycosylated (tetra-saccharide moiety i.e. lycotetrose) toproduce dehydrotomatine and α-tomatine, respectively. Thus, the SGApathway branches at dehydrotomatidine for either formation of tomatidinederived SGAs or glycosylated dehydrotomatine derivatives. Notably,dehydrotomatidine and tomatidine are only different in their structuresby the presence or absence of the double bond at the C-5 position. Theconversion of dehydrotomatidine to tomatidine was hypothesized in thepast as a single reaction catalyzed by a hypothetical hydrogenase. Inmost tomato plant tissues, the relative portion of dehydrotomatine ascompared to α-tomatine ranges from ˜2.5-˜10%. As tomato fruit maturesand reaches to the red stage, the entire pool of α-tomatine anddehydrotomatine is largely being converted to esculeosides (major SGAs)and dehydroesculeosides (minor SGAs), respectively.

In cultivated potato, α-chaconine and α-solanine are the major SGAssharing the same aglycone, solanidine (in which a C-5,6 double bond ispresent) and possess chacotriose and solatriose moieties, respectively.As there is no demissidine or demissine detected in cultivated potatoes,it was suggested that a hydrogenase enzyme able to convert solanidine todemissidine is lacking in these species. Several wild potato species(e.g. S. demissum, S. chacoense, S. commersonii) and their somatichybrids (S. brevidens×S. tuberosum), predicted to contain an activehydrogenase, do produce demissidine or its glycosylated form, demissinebeing one of their major SGAs.

In eggplant, α-solamargine and α-solasonine are the most abundant SGAsderived from the solasodine aglycone (in which a C-5,6 double bond ispresent); while some wild solanum species, e.g. S. dulcamara producesoladulcidine or its glycosylated forms, soladulcine A and β-soladulcine(C-5,6 double bond is absent), as major SGAs from the solasodineaglycone.

In addition to SGAs, many Solanum species (e.g. eggplant) also producecholesterol-derived unsaturated or saturated steroidal saponins.Unsaturated and saturated steroidal saponins are widespread in the plantkingdom, especially among monocots, e.g. the Agavaceae (e.g., agave andyucca), Asparagaceae (e.g., asparagus), Dioscoreaceae and Liliaceaefamilies. Similar to SGAs, steroidal saponins are highly diverse instructures and could be either saturated (e.g. sarasapogenin) orunsaturated (e.g. diosgenin) in the C-5,6 position.

Cholesterol, the main sterol produced by all animals, serves as a keybuilding block in the biosynthesis of SGAs. An array of tomato andpotato GLYCOALKALOIDMETABOLISM (GAME) genes participating in core SGAbiosynthesis starting from cholesterol were reported in recent years.The tomato SGAs biosynthetic pathway can be divided into two main parts.In the first, the SA aglycone is formed from cholesterol by the likelyaction of the GAME6, GAME8, GAME11, GAME4 and GAME12 enzymes. The secondpart results in the generation of SGA through the action ofUDP-glycosyltransferases (UGTs): GAME1, GAME2, GAME17 and GAME18 intomato, and STEROL ALKALOID GLYCOSYL TRANSFERASE1 (SGT1), SGT2 and SGT3in potato.

The demand for higher food quantities and food with improved qualitycontinues to increase. Improved nutritional qualities as well as removalof antinutritional traits are both of high demand. In the course of cropdomestication, levels of anti-nutrients were reduced by breeding.However, Solanaceous crop plants still contain significant amount ofantinutritional substances, particularly steroidal glycoalkaloids.

Alternatively, the ability to manipulate the synthesis of these SGAswould provide the means to develop, through classical breeding orgenetic engineering, crops with modified levels and composition of SGAs,conferring on the plant an endogenous chemical barrier against a broadrange of insects and other pathogens.

In addition, there is a demand both for plant-based cholesterols and,conversely, for plants with increased levels of phytocholesterols orother phytosterols.

Thus, there is a demand for, and would be highly advantageous to havemeans and method for controlling the production of steroidal alkaloidsin Solanaceous plants for obtaining high quality non-toxic food productsas well as for the production of steroidal alkaloids and phytosterolswith beneficial, particularly therapeutic, effects.

SUMMARY OF DISCLOSURE

The present invention relates to key genes and enzymes in thebiosynthesis pathway converting cholesterol to steroidal glycoalkaloids(SGA), useful for modulating the expression of steroidal alkaloids andin plants. Particularly, the present invention relates to transgenicSolanaceous plants with reduced content of antinutritional alkaloids.

The present invention is based in part on the unexpected discovery thatthe biosynthesis of SGAs in Solanaceous plant involves an array ofgenes, wherein 5-7 of the genes (depending on the plant species) areclustered on chromosome 7 and additional two genes are placed adjacentin a duplicated genomic region on chromosome 12. Several regulatorygenes, including transcription factors were found to be co-expressedwith the clustered genes. Modulating the expression of particular geneswithin the array enabled strict control of the production of steroidalalkaloids and glycosylated derivatives thereof. Unexpectedly, modulatingthe expression of a single gene or transcription factor resulted insignificant elevation/reduction in the content of steroidal alkaloids(e.g., solanine and/or chaconine in potato), in tomato, potato andeggplant plants, of α-tomatine in tomato plants, of cholesterol intomato plants. Particularly, the present invention now shows thatmodulating a single transcription factor, designated hereinGAME9-transcription factor resulted in strict control on the productionof steroidal glycoalkaloids (SGAs) in potato tuber peels. Particularly,the present invention now shows that modulating a single protein,designated herein GAME15 (the product of a cellulose synthase likegene), resulted in strict control on the production of steroidalglycoalkaloids (SGAs) and steroidal saponins in tomatoes, potatoes, andeggplants. Inhibiting the expression of a gene encoding2-oxoglutarate-dependent dioxygenase (GAME11) resulted in a significantreduction in α-tomatine level and accumulation of several phytosterols,including cholesterol, cholestanol, and any of their modifiedglycosylated derivatives, steroidal saponins in tomato plants.Inhibiting the expression of a gene encoding cellulose synthase likeprotein (GAME15) resulted in a significant reduction in levels ofα-tomatine and downstream SGAs (including esculeosides) in tomato plantsand an accumulation of cholesterol (a precursor for SGAs) in tomatoplants. In potato, silencing of GAME15 resulted in significantreductions in α-chaconine and α-solanine and in accumulation of acholesterol pool. According to one aspect, the present inventionprovides a genetically modified or gene edited plant comprising at leastone cell having altered expression of at least one gene selected fromthe group consisting of a gene encoding a cellulose synthase likeprotein ((GAME15), wherein the genetically modified or gene edited planthas an altered content of at least one steroidal alkaloid or aglycosylated derivative thereof compared to a corresponding unmodifiedor unedited plant.

According to one aspect, the present invention provides a geneticallymodified plant comprising at least one cell having altered expression ofat least one gene selected from the group consisting of a gene encodingat least one cellulose synthase like protein compared to its expressionin a corresponding unmodified plant, wherein the genetically modifiedplant has an altered content of at least one steroidal alkaloid or aglycosylated derivative thereof compared to the corresponding unmodifiedplant.

According to certain embodiments, expression of the gene encoding the atleast one cellulose synthase like protein is reduced compared to itsexpression in the corresponding unmodified plant, thereby thegenetically modified plant comprises reduced content at least onesteroidal alkaloid or a glycosylated derivative thereof compared to thecorresponding unmodified plant. According to other embodiments,expression of the gene encoding the at least one cellulose synthase likeprotein is elevated compared to its expression in the correspondingunmodified plant, thereby the genetically modified plant compriseselevated content at least one steroidal alkaloid or a glycosylatedderivative thereof compared to the corresponding unmodified plant.

According to one aspect, the present invention provides method ofreducing the content of at least one steroidal alkaloid or aglycosylated derivative thereof in a modified plant, the methodcomprising (a) transforming at least one plant cell with at least onesilencing molecule targeted to a nucleic acid sequence encoding at leastone protein comprising a cellulose synthase like protein; or (b)mutagenizing at least one gene or a combination of genes, the genesencoding at least one protein selected from the group consisting ofcellulose synthase like proteins, wherein the mutagenesis comprisesintroduction of one or more point mutations into the gene, or genomeediting, or use of a bacterial CRISPR/CAS system, or a combinationthereof, wherein expression of the gene encoding the at least onecellulose synthase like protein is reduced in the modified plantcompared to its expression in a corresponding unmodified plant, therebythe modified plant comprises reduced content at least one steroidalalkaloid or a glycosylated derivative thereof compared to thecorresponding unmodified plant.

According to one aspect, the present invention provides a method ofproducing at least one phytosterol in a modified plant, the methodcomprising (a) transforming at least one plant cell with at least onesilencing molecule targeted to a nucleic acid sequence encoding at leastone protein comprising a cellulose synthase like factor; or (b)mutagenizing at least one gene or a combination of genes, the genesencoding at least one protein selected from the group consisting ofcellulose synthase like factors, wherein the mutagenesis comprisesintroduction of one or more point mutations into the gene, or genomeediting, or use of a bacterial CRISPR/CAS system, or a combinationthereof, wherein expression of the gene encoding the at least onecellulose synthase like protein is reduced in the modified plantcompared to its expression in a corresponding unmodified plant, therebythe modified plant comprises reduced content at least one steroidalalkaloid or a glycosylated derivative thereof compared to thecorresponding unmodified plant.

According to certain embodiments, the plant is a transgenic plantcomprising at least one cell comprising at least one transcribablepolynucleotide encoding at least one protein comprising a cellulosesynthase like protein. According to certain embodiments, thetranscribable polynucleotide comprises the nucleic acid sequence setforth in any one of SEQ ID NOS: 32, 34, 36, 38, 40, or 42.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the proposed biosynthetic pathway of steroidalglycoalkaloids in the triterpenoid biosynthetic pathway in Solanaceousplant from cholesterol toward α-tomatine. Dashed and solid arrowsrepresent multiple or single enzymatic reactions in the pathway,respectively.

FIG. 2 summarizes the coexpression analysis of steroidalalkaloid-associated genes in Solanaceous plants. Shared homologs ofcoexpressed genes for ‘baits’ from tomato (SlGAME1 and SlGAME4) andpotato (StSGT1 and StGAME4). Continuous (r-value>0.8) and dashed(r-value>0.63) lines connect coexpressed genes. *, located in the tomatoor potato chromosome 7 cluster. St, Solanum tuberosum; Sl, S.lycopersicum. Background of gene names corresponds to bait they werefound to be coexpressed with (legend above). SP, serine proteinase; PI,proteinase inhibitor; UPL, ubiquitin protein ligase; ELP, extensin-likeprotein; PK, protein kinase; SR, sterol reductase; RL, receptor-like.

FIG. 3 presents schematic map of genes identified in the duplicatedgenomic regions in tomato and potato and their coexpression.Coexpression with GAME1/SGT1 (chromosome 7) and GAME4 (chromosome 12) asbaits in either potato or tomato are presented in a form of a heatmap(Tables 3-6). Specific gene families are indicated by dark arrows whilemembers of other gene families are in white arrows.

FIGS. 4A-4H shows functional analysis of tomato GAME genes. (FIG. 4A)GAME8-silenced transgenic (RNAi) leaves accumulated22-(R)-hydroxycholesterol compared to wild type. (FIG. 4B) An array ofcholestanol-type steroidal saponins (STSs) accumulates in GAME11VIGS-silenced leaves. (FIG. 4C) An STS (m/z=753.4) accumulates in GAME12VIGS-leaves. (FIG. 4D) Tomatidine, the steroidal alkaloid aglycone,accumulates in GAME1-silenced transgenic leaves. (FIGS. 4E to 4H) Enzymeactivity assays of the 4 recombinant tomato GAME glycosyltransferases.

FIGS. 5A-5D show solanine/chaconine levels in peels of tuber of potatoplant lines with altered expression of GAME9 compared to wild typeplants. Solanine (FIG. 5A) and chaconine (FIG. 5B) level in tubers ofGAME9 silenced plant; Solanine (FIG. 5C) and chaconine (FIG. 5D) levelsin tubers of GAME9 overexpressing plants.

FIG. 6 shows solanine/chaconine levels in leaves of potato plant lineswith either silenced (RNAi) or overexpressed (OX) GAME9 compared to wildtype plants.

FIG. 7 shows tomatine levels in leaves of tomato plant lines with eithersilenced (RNAi, line 5871) or overexpressed (OX, line 5879) GAME9compared to wild type plants.

FIGS. 8A-8D show the effect of silencing of GAME11 dioxygenase intomato. (FIG. 8A) α-tomatine levels in leaves (m/z=1034.5) (FIG. 8B)cholestanol-type steroidal saponins (STS) in leaves (m/z=1331.6, 1333.6,1199.6, 1201.6 (major saponins)). (FIG. 8C) MS/MS spectrum of m/z=1331.6(at 19.28 min.). (FIG. 8D) The fragmentation patterns of the saponineluted at 19.28 min. and accumulating in GAME11-silenced leaves.Corresponding mass signals are marked with an asterisk on the MS/MSchromatogram in FIG. 8C.

FIGS. 9A-9E show metabolites extracted from GAME18-silenced mature greentomato fruit. Peaks of newly accumulating compounds corresponding to theγ-tomatine standard (m/z=740.5) (FIGS. 9A-9C), and γ-tomatine pentoside(m/z=872.5) (FIGS. 9D-9E) are shown.

FIGS. 10A-10D show the effect of silencing of GAME12 transaminase intomato. (FIG. 10A) accumulation of a furastanol-type STS. (FIGS.10B-10C) GAME12-silenced leaves accumulate an STS (m/z=753.4), while itexists in only minor quantities in WT leaf. (FIG. 10D) MS/MS spectrum ofm/z=753.4 at 19.71 min. with interpretation of the fragments.

FIGS. 11A-11D show the effect silencing of GAME8 in tomato plants.GAME8-silenced leaves accumulated 22-(S) and -(R)-cholesterol (FIG.11A). Chromatograms (mass range 172.5-173.5) acquired via EI-GC/MS, MSspectra and structures (tri-methyl-silyl derivatives) of the compoundsare shown. Commercial standards of 22-(R)-(FIG. 11B) and22-(S)-cholesterol (FIG. 11C) were used to verify the putativeidentification. (FIG. 11D) GAME8-silenced line accumulates both isomersin comparison to WT (Q).

FIG. 12 shows the phylogenetic tree of GAME genes in the plant CYP450protein family. The numbers on the branches indicate the fraction ofbootstrap iterations supporting each node.

FIG. 13 shows a proposed expanded biosynthetic pathway in Solanaceousplants from Cycloartenol (Part I), through Cholesterol (Part II),through Tomatidine (Part III), through Steroidal Glycoalkaloidsincluding α-tomatine to Lycoperosides/Esculeoside (Part IV). Dashedarrows represent multiple enzymatic reactions in the pathway.

FIGS. 14A-14C show an overview of SGA biosynthesis in (FIG. 14A) tomato,(FIG. 14B) potato, and (FIG. 14C) eggplant.

FIGS. 15A-15C show major SGA levels in (FIG. 15A) leaves and (FIG. 15B)green fruit and (FIG. 15C) red fruit of wild type (non-transformed) andGAME15-RNAi tomato lines determined by LC-MS, #21, #22 and #23 are threeindependent GAME15-RNAi transgenic tomato lines. Values indicate meansof three biological replicates±standard error. Asterisks indicatesignificant changes from wild-type samples as calculated by a Student'st-test (*P-value<0.05; **P-value<0.01; ***P-value<0.001).

FIG. 16 shows levels of α-solanine and α-chaconine in leaves ofGAME15-RNAi lines as determined by LC-MS. #1, #2 and #3 are threeindependent GAME15i transgenic potato lines. Values representmean±standard error n=3). Student's t-test was used to assess whetherthe transgenic lines significantly differ from wild-type plants:(*P-value<0.05; **P-value<0.01; ***P-value<0.001).

FIG. 17 shows the cholesterol content of tomato leaves derived fromGAME15 silenced plants. Values represent mean of three biologicalreplicates±standard error. Asterisks indicate significant changes inleaves of the three independent transgenes (#21, #22 and #23) ascompared to wild-type leaves (i.e. non-transformed) calculated by aStudent's t-test (*P-value<0.05; **P-value<0.01; ***P-value<0.001).Epicholesterol was used as an internal standard in sample preparationsand relative cholesterol level is expressed as ratios of cholesterolpeak areas in sample compared to internal standard. The analysis wasperformed using GC-MS.

DETAILED DESCRIPTION

According to one aspect, the present invention provides a geneticallymodified plant comprising at least one cell having altered expression ofat least one gene selected from the group consisting of a gene encodingat least one cellulose synthase like protein compared to its expressionin a corresponding unmodified plant, wherein the genetically modifiedplant has an altered content of at least one steroidal alkaloid or aglycosylated derivative thereof compared to the corresponding unmodifiedplant.

According to certain embodiments, the cellulose synthase like protein isa GAME15 protein. According to certain embodiments, the amino acidsequence of the cellulose synthase like protein of the correspondingunmodified plant comprises the sequence set for cellulose synthase likeprotein is at least 80% homologous to the amino acid sequence set forthin any one of SEQ ID NOS: 33, 35, 37, 39, 42, or 43. According tocertain embodiments, the polynucleotide encoding the cellulose synthaselike protein of the corresponding unmodified plant comprises the nucleicacid sequence set forth in any one of SEQ ID NOS: 32, 34, 36, 38, 40, or42.

According to certain embodiments, expression of the at least one gene orany combination thereof is altered, the altering comprising mutagenizingthe at least one gene, wherein the mutagenesis comprises introduction ofone or more point mutations, or genome editing, or use of a bacterialCRISPR/CAS system, or a combination thereof.

According to certain embodiments, expression of the gene encoding the atleast one cellulose synthase like protein is reduced compared to itsexpression in the corresponding unmodified plant, thereby thegenetically modified plant comprises reduced content at least onesteroidal alkaloid or a glycosylated derivative thereof compared to thecorresponding unmodified plant.

According to certain embodiments, the genetically modified plant is atransgenic plant comprising at least one cell comprising at least onesilencing molecule targeted to a polynucleotide encoding at least onecellulose synthase like protein. According to certain embodiments, thetransgenic plant comprises a polynucleotide encoding a cellulosesynthase like protein, wherein expression of the polynucleotide isselectively silenced, repressed, or reduced. According to certainembodiments, the transgenic plant comprises a polynucleotide encoding acellulose synthase like protein, wherein the polynucleotide has beenselectively edited by deletion, insertion, or modification to silence,repress, or reduce expression thereof, or wherein the geneticallymodified plant is a progeny of the gene edited plant.

According to certain embodiments, the transgenic plant comprises atleast one cell comprising at least one silencing molecule targeted to aGAME15 gene.

According to certain embodiments, the transgenic plant comprises atleast one cell comprising at least one silencing molecule targeted tothe nucleic acid sequence set forth in any one of SEQ ID NOS: 32, 34,36, 38, 40, or 42. According to certain embodiments, the silencingmolecule is selected from the group consisting of an RNA interferencemolecule and an antisense molecule, or wherein the silencing molecule isa component of a viral induced gene silencing system. According tocertain embodiments, the silencing molecule comprises a polynucleotidehaving a nucleic acid sequence substantially complementary to a regionof the GAME15 gene having the nucleic acid sequence set forth in any oneSEQ ID NOS: 32, 34, 36, 38, 40, or 42 or a complementary sequencethereof. According to certain embodiments, the silencing molecule istargeted to a GAME15 fragment having the nucleic acid sequence set forthin SEQ ID NO: 44 or a complementary sequence thereof. According tocertain embodiments, the silencing molecule is targeted to a GAME15fragment having the nucleic acid sequence set forth in SEQ ID NO: 45 ora complementary sequence thereof. According to certain embodiments, thesilencing molecule is targeted to a GAME15 fragment having the nucleicacid sequence set forth in SEQ ID NO: 46 or a complementary sequencethereof.

According to certain embodiments, the genetically modified plant is aSolanaceae plant having a reduced content of at least one steroidalglycoalkaloid selected from the group consisting of α-tomatine,tomatidine, α-chaconine, α-solanine, α-solasonine, α-solmargine, andderivatives thereof, compared to a corresponding unmodified plant.According to certain embodiments, the genetically modified plant furthercomprises an elevated content of a phytosterol or a derivative thereof,a cholesterol or a derivative thereof, a phytocholesterol or aderivative thereof, a cholestenol or a derivative thereof, aphytocholestanol or a derivative thereof, or a steroidal saponin or aderivative thereof compared to a corresponding unmodified plant.

According to certain embodiments, the plant is a Solanaceae plantselected from the group consisting of tomato, potato, eggplant, andpepper. According to certain embodiments, the plant is a tomato plantcomprising a reduced content of α-tomatine, tomatidine, or derivativesthereof. According to certain embodiments, the plant is a tomato plantcomprising an elevated content of a phytosterol, a phytocholesterol orcholesterol, a phytocholestenol or cholestenol, a steroidal saponin, orderivative thereof. According to certain embodiments, the plant is apotato plant comprising a reduced content of α-chaconine, α-solanine, orderivatives thereof. According to certain embodiments, the plant is aneggplant plant comprising a reduced content of α-solasonine,α-solamargine, or derivatives thereof.

According to other certain embodiments, expression of the gene encodingthe at least one cellulose synthase like protein is elevated compared toits expression in the corresponding unmodified plant, thereby thegenetically modified plant comprises elevated content at least onesteroidal alkaloid or a glycosylated derivative thereof compared to thecorresponding unmodified plant. According to certain embodiments, thetransgenic plant comprises a polynucleotide encoding a cellulosesynthase like protein, wherein expression of the polynucleotide isselectively increased. According to certain embodiments, the transgenicplant comprising at least one cell comprising at least one transcribablepolynucleotide encoding at least one protein selected from the groupconsisting of at least one a cellulose synthase like protein. Accordingto certain embodiments, the cellulose synthase like protein is a GAME15protein. According to certain embodiments, the transcribablepolynucleotide comprises the nucleic acid sequence set forth in any oneof SEQ ID NOS: 32, 34, 36, 38, 40, or 42. According to certainembodiments, the genetically modified plant is a Solanaceae plant havingan elevated content of at least one steroidal glycoalkaloid selectedfrom the group consisting of α-tomatine, tomatidine, α-chaconine,α-solanine, α-solasonine, α-solmargine, and derivatives thereof,compared to a corresponding unmodified plant. According to certainembodiments, the genetically modified plant further comprises a reducedcontent of a phytosterol or a derivative thereof, a cholesterol or aderivative thereof, a phytocholesterol or a derivative thereof, acholestenol or a derivative thereof, a phytocholestanol or a derivativethereof, or a steroidal saponin or a derivative thereof compared to acorresponding unmodified plant. According to certain embodiments, theplant is a Solanaceae plant selected from the group consisting oftomato, potato, eggplant, and pepper.

According to one aspect, the present invention provides a method ofreducing the content of at least one steroidal alkaloid or aglycosylated derivative thereof in a modified plant, the methodcomprising (a) transforming at least one plant cell with at least onesilencing molecule targeted to a nucleic acid sequence encoding at leastone protein comprising a cellulose synthase like protein; or (b)mutagenizing at least one gene or a combination of genes, the genesencoding at least one protein selected from the group consisting ofcellulose synthase like proteins, wherein the mutagenesis comprisesintroduction of one or more point mutations into the gene, or genomeediting, or use of a bacterial CRISPR/CAS system, or a combinationthereof wherein expression of the gene encoding the at least onecellulose synthase like protein is reduced in the modified plantcompared to its expression in a corresponding unmodified plant, therebythe modified plant comprises reduced content at least one steroidalalkaloid or a glycosylated derivative thereof compared to thecorresponding unmodified plant.

According to certain embodiments, the cellulose synthase like protein isa GAME15 protein. According to certain embodiments, the amino acidsequence of the cellulose synthase like protein of the correspondingunmodified plant comprises the sequence set for cellulose synthase likeprotein is at least 80% homologous to the amino acid sequence set forthin any one of SEQ ID NOS: 33, 35, 37, 39, 42, or 43. According tocertain embodiments, wherein the polynucleotide encoding the cellulosesynthase like protein of the corresponding unmodified plant comprisesthe nucleic acid sequence set forth in any one of SEQ ID NOS: 32, 34,36, 38, 40, or 42.

According to certain embodiments, the silencing molecule is targeted toa GAME15 fragment having the nucleic acid sequence set forth in SEQ IDNO: 44 or a complementary sequence thereof. According to certainembodiments, the silencing molecule is targeted to a GAME15 fragmenthaving the nucleic acid sequence set forth in SEQ ID NO: 45 or acomplementary sequence thereof. According to certain embodiments, thesilencing molecule is targeted to a GAME15 fragment having the nucleicacid sequence set forth in SEQ ID NO: 46 or a complementary sequencethereof.

According to certain embodiments, the modified plant is a Solanaceaeplant having a reduced content of at least one steroidal glycoalkaloidselected from the group consisting of α-tomatine, tomatidine,α-chaconine, α-solanine, α-solasonine, α-solmargine, and derivativesthereof, compared to the corresponding unmodified plant.

According to certain embodiments, the modified plant further comprisesan elevated content of a phytosterol or a derivative thereof acholesterol or a derivative thereof, a phytocholesterol or a derivativethereof, a cholestenol or a derivative thereof, a phytocholestanol or aderivative thereof, or a steroidal saponin or a derivative thereofcompared to a corresponding unmodified plant.

According to certain embodiments, the modified plant is a Solanaceaeplant selected from the group consisting of tomato, potato, eggplant,and pepper. According to certain embodiments, the plant is a tomatoplant comprising a reduced content of α-tomatine, tomatidine, orderivatives thereof. According to certain embodiments, the plant is atomato plant comprising an elevated content of a phytosterol, aphytocholesterol or cholesterol, a phytocholestenol or cholestenol, asteroidal saponin, or derivative thereof. According to certainembodiments, the plant is a potato plant comprising a reduced content ofα-chaconine, α-solanine, or derivatives thereof. According to certainembodiments, the plant is an eggplant plant comprising a reduced contentof α-solasonine, α-solamargine, or derivatives thereof.

According to one aspect, the present invention provides a method ofproducing at least one phytosterol in a modified plant, the methodcomprising (a) transforming at least one plant cell with at least onesilencing molecule targeted to a nucleic acid sequence encoding at leastone protein comprising a cellulose synthase like factor; or (b)mutagenizing at least one gene or a combination of genes, the genesencoding at least one protein selected from the group consisting ofcellulose synthase like factors, wherein the mutagenesis comprisesintroduction of one or more point mutations into the gene, or genomeediting, or use of a bacterial CRISPR/CAS system, or a combinationthereof, wherein expression of the gene encoding the at least onecellulose synthase like protein is reduced in the modified plantcompared to its expression in a corresponding unmodified plant, therebythe modified plant comprises reduced content at least one steroidalalkaloid or a glycosylated derivative thereof compared to thecorresponding unmodified plant.

According to certain embodiments, the cellulose synthase like protein isa GAME15 protein. According to certain embodiments, the amino acidsequence of the cellulose synthase like protein of a correspondingunmodified plant comprises the sequence set for cellulose synthase likeprotein is at least 80% homologous to the amino acid sequence set forthin any one of SEQ ID NOS: 33, 35, 37, 39, 42, or 43. According tocertain embodiments, the polynucleotide encoding the cellulose synthaselike protein of a corresponding unmodified plant comprises the nucleicacid sequence set forth in any one of SEQ ID NOS: 32, 34, 36, 38, 40, or42.

According to certain embodiments, the method further comprises purifyingthe phytosterol extracted from the transformed plant. According tocertain embodiments, the phytosterol comprises phytocholesterol.

According to certain embodiments, the silencing molecule is targeted toa GAME15 fragment having the nucleic acid sequence set forth in SEQ IDNO: 44 or a complementary sequence thereof. According to certainembodiments, the silencing molecule is targeted to a GAME15 fragmenthaving the nucleic acid sequence set forth in SEQ ID NO: 45 or acomplementary sequence thereof. According to certain embodiments, thesilencing molecule is targeted to a GAME15 fragment having the nucleicacid sequence set forth in SEQ ID NO: 46 or a complementary sequencethereof.

According to certain embodiments, the modified plant further comprisesan elevated content of a phytosterol or a derivative thereof, acholesterol or a derivative thereof, a phytocholesterol or a derivativethereof, a cholestenol or a derivative thereof, a phytocholestanol or aderivative thereof, or a steroidal saponin or a derivative thereofcompared to a corresponding unmodified plant.

According to certain embodiments, the modified plant is a Solanaceaeplant. According to certain embodiments, the Solanaceae plant isselected from the group consisting of tomato, potato, eggplant, andpepper.

It is to be understood that inhibiting the expression of the at leastone gene or combination thereof may be achieved by various means, all ofwhich are explicitly encompassed within the scope of present invention.According to certain embodiments, inhibiting the expression of GAME15can be affected at the genomic and/or the transcript level using avariety of molecules that interfere with transcription and/ortranslation including, but not limited to, antisense, siRNA, Ribozyme,or DNAzyme molecules. Inserting a mutation to the at least one gene,including deletions, insertions, site specific mutations, zinc-fingernucleases and the like can be also used, as long as the mutation resultsin down-regulation of the gene expression. According to otherembodiments, expression is inhibited at the protein level usingantagonists, enzymes that cleave the polypeptide and the like.

According to certain exemplary embodiments, the genetically modified orgene edited plant is a transgenic plant comprising at least one cellcomprising at least one silencing molecule targeted to a GAME15 gene.Each possibility represents a separate embodiment of the presentinvention. According to some embodiments, the at least one silencingmolecule is selected from the group consisting of RNA interferencemolecule and anti sense molecule. According to these embodiments, thetransgenic plant comprises reduced content of at least one steroidalalkaloid or glycosylated derivative thereof, or of at least onesteroidal saponin or glycosylated derivative thereof, compared tonon-transgenic plant. According to certain embodiments, the at least onesteroidal alkaloid is steroidal glycoalkaloid. According to certainexemplary embodiments, the steroidal glycoalkaloid is selected from thegroup consisting of α-solanine, α-chaconine, α-solmargine, α-solasonine,α-tomatine, tomatidine and derivatives thereof. According to certainembodiments, the transgenic plant comprises reduced content of at leastone downstream steroidal alkaloid or glycosylated derivative thereofcompared to non-transgenic plant. According to certain exemplaryembodiments, the downstream steroidal glycoalkaloid is selected from thegroup consisting of esculeosides. According to certain embodiments, thetransgenic plant comprises increased content of at least onephytosterol. In some embodiments, the phytosterol is a phytocholesterol,a cholesterol, or a cholestanol. According to some embodiments, thetransgenic plant comprises a plurality of cells comprising the silencingmolecule targeted to at least one GAME15 gene. According to additionalembodiments, the majority of the plant cells comprise the silencingmolecule.

The silencing molecule target to at least one GAME15 can be designed asis known to a person skilled in the art. According to certainembodiments, the silencing molecule comprises a polynucleotide having anucleic acid sequence substantially complementary to a region of theGAME15, gene or to a complementary sequence of GAME15, e.g., having thenucleic acids sequence set forth in any one of SEQ ID NOS: 44 to 46.Each possibility represents a separate embodiment of the presentinvention.

According to certain exemplary embodiments, the silencing molecule istargeted to a GAME15 fragment having the nucleic acids sequence setforth in SEQ ID NOS: 44 to 46 or a complementary sequence thereof.

According to certain additional embodiments, the silencing moleculecomprises a polynucleotide having a nucleic acid sequence substantiallycomplementary to a region of the GAME15 gene or a complementary sequencethereof, having the nucleic acids sequence set forth in any one of SEQID NOS: 44 to 46. Each possibility represents a separate embodiment ofthe present invention.

According to certain embodiments, the silencing molecule is an antisenseRNA.

According to certain exemplary embodiments, the silencing molecule is anRNA interference (RNAi) molecule. According to some embodiments, thesilencing molecule is a double-stranded (ds)RNA molecule. According tocertain embodiments, the first and the second polynucleotides areseparated by a spacer. According to exemplary embodiments, the spacersequence is an intron. According to yet further embodiments, theexpression of the first and the second polynucleotides is derived fromone promoter. According to other embodiments, expression of the firstand the second polynucleotides are derived from two promoters; thepromoters can be identical or different. Each possibility represents aseparate embodiment of the present invention.

According certain exemplary embodiments, the dsRNA is targeted toGAME15, said dsRNA molecule comprises a first polynucleotide and asecond polynucleotide having a nucleic acid sequence complementary tosaid first polynucleotide.

According to certain embodiments, the transgenic tomato plant furthercomprises elevated amounts of steroidal saponins. According to certainembodiments, the steroidal saponin is a cholesterol-derived saponin.Each possibility represents a separate embodiment of the presentinvention.

Overexpression of the at least one gene can be obtained by any method asis known to a person skilled in the art. According to certainembodiments, the present invention provides a transgenic plantcomprising at least one cell comprising at least one transcribablepolynucleotide encoding at least one GAME15 protein, wherein thetransgenic plant comprises elevated content of at least one steroidalalkaloid or a glycosylated derivative thereof compared to acorresponding non-transgenic plant or reduced content of at least onephytosterol.

According to some embodiments, the polynucleotides of the presentinvention are incorporated in a DNA construct enabling their expressionin the plant cell. DNA constructs suitable for use in plants are knownto a person skilled in the art. According to one embodiment, the DNAconstruct comprises at least one expression regulating element selectedfrom the group consisting of a promoter, an enhancer, an origin ofreplication, a transcription termination sequence, a polyadenylationsignal and the like.

The DNA constructs of the present invention are designed according tothe results to be achieved. In crop plants, reduction of toxic steroidalglycoalkaloids is desired in the edible parts of the plant, including,for example, fruit and tubers. On the other hand, enriching the contentof toxic steroidal glycoalkaloids in non-edible roots and leavescontributes to the resistance of the plant against a broad range ofpathogens. Plants overexpressing the steroidal glycoalkaloids can beused for producing them for the pharmaceutical industry.

According to certain embodiments, the DNA construct comprises apromoter. The promoter can be constitutive, induced or tissue specificas is known in the art. Optionally, the DNA construct further comprisesa selectable marker, enabling the convenient selection of thetransformed cell/tissue. Additionally, or alternatively, a reporter genecan be incorporated into the construct, so as to enable selection oftransformed cells or tissue expressing the reporter gene.

Suspensions of genetically modified or gene edited cells and tissuecultures derived from the genetically modified or gene edited cells arealso encompassed within the scope of the present invention. The cellsuspension and tissue cultures can be used for the production of desiredsteroidal glycoalkaloids and, which are then extracted from the cells orthe growth medium. Alternatively, the genetically modified or geneedited cells and/or tissue culture are used for regenerating atransgenic plant having modified or gene edited expression of GAME15,therefore having modified content of steroidal glycoalkaloids.

The present invention further encompasses seeds of the geneticallymodified or gene edited plant, wherein plants grown from said seeds havealtered expression of GAME15 compared to plants grown from correspondingunmodified or unedited seeds, thereby having an altered content of atleast one steroidal glycoalkaloid.

Genetically Modified Plants & Gene Edited Plants

Disclosed herein are genetically modified plants and gene edited plants,wherein expression of key genes in the steroidal glycoalkaloidsmetabolic pathway (biosynthesis pathway of steroidal alkaloids andglycosylated derivatives thereof) have been altered. Altering theexpression of these genes results in concomitant alteration in thesteroidal alkaloid profile. Changing the production level of steroidalalkaloid can result in improved plants comprising elevated content ofsteroidal alkaloids having increased resistance to pathogens, or plantshaving a reduced content of these secondary compounds in the plantedible parts and thus producing improved crops, wherein the improvedcrop has reduced or eliminated anti-nutritional content. Alternatively,or additionally, controlling the expression of genes disclosed hereinmay be used for the production of desired steroidal alkaloids orplant-based cholesterol for further use, for example in thepharmaceutical industry. In particular, disclosed herein are the meansand methods for producing crop plants of the Solanaceae family that aredevoid of toxic amounts of deleterious steroidal alkaloids typicallypresent in edible parts of these plants. The plants disclosed herein arethus of significant nutritional and commercial value.

Disclosed herein are an array of co-expressed genes that participate inthe biosynthesis pathway of steroidal alkaloids. The present inventionfurther discloses key genes in this pathway, altering the expression ofwhich result in concomitant alteration in the steroidal alkaloidprofile. Changing the production level of steroidal alkaloid can resultin an improved plant comprising elevated content of steroidal alkaloidshaving increased resistance to pathogens, or plants having a reducedcontent of these secondary compounds in the plant edible parts and thusproducing improved crops. Alternatively, or additionally, controllingthe expression of genes revealed in the present invention can be usedfor the production of desired steroidal alkaloids or plant-basedcholesterol for further use, for example in the pharmaceutical industry.In particular, the present invention discloses means and methods forproducing crop plants of the genus Solanum that are devoid of toxicamounts of deleterious steroidal alkaloids typically present in edibleparts of these plants. The plants of the present invention are thus ofsignificant nutritional and commercial value.

Definitions

As used herein, the term “Solanaceous” refers to a plant of the genusSolanum.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of RNA or apolypeptide. A polypeptide can be encoded by a full-length codingsequence or by any part thereof. The term “parts thereof” when used inreference to a gene refers to fragments of that gene. The fragments mayrange in size from a few nucleotides to the entire gene sequence minusone nucleotide. Thus, “a nucleic acid sequence comprising at least apart of a gene” may comprise fragments of the gene or the entire gene.

The term “gene” optionally also encompasses the coding regions of astructural gene and includes sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of about 1 kb on eitherend such that the gene corresponds to the length of the full-lengthmRNA. The sequences which are located 5′ of the coding region and whichare present on the mRNA are referred to as 5′ non-translated sequences.The sequences which are located 3′ or downstream of the coding regionand which are present on the mRNA are referred to as 3′ non-translatedsequences.

One of ordinary skill in the art would appreciate that the term “gene”may encompass a nucleic acid (e.g., DNA or RNA) sequence that comprisescoding sequences necessary for the production of RNA or a polypeptide. Apolypeptide can be encoded by a full-length coding sequence or by anypart thereof. The term “parts thereof” when used in reference to a generefers to fragments of that gene. The fragments may range in size from afew nucleotides to the entire gene sequence minus one nucleotide. Thus,“a nucleic acid sequence comprising at least a part of a gene” maycomprise fragments of the gene or the entire gene.

The skilled artisan would appreciate that the term “gene” optionallyalso encompasses the coding regions of a structural gene and includessequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb on either end such that the genecorresponds to the length of the full-length mRNA. The sequences whichare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ non-translated sequences. The sequences which arelocated 3′ or downstream of the coding region and which are present onthe mRNA are referred to as 3′ non-translated sequences.

In one embodiment, a gene comprises DNA sequence comprising upstream anddownstream regions, as well as the coding region, which comprises exonsand any intervening introns of the gene. In some embodiments, upstreamand downstream regions comprise non-coding regulatory regions. In someembodiments, upstream and downstream regions comprise regulatorysequences, for example but not limited to promoters, enhancers, andsilencers. Non-limiting examples of regulatory sequences include, butare not limited to, AGGA box, TATA box, Inr, DPE, ZmUbi1, PvUbi1,PvUbi2, CaMV, 35S, OsAct1, zE19, E8, TA29, A9, pDJ3S, B33, PAT1, alcA,G-box, ABRE, DRE, and PCNA. Regulatory regions, may in some embodiments,increase or decrease the expression of specific genes within a plantdescribed herein.

In another embodiment, a gene comprises the coding regions of the gene,which comprises exons and any intervening introns of the gene. Inanother embodiment, a gene comprises its regulatory sequences. Inanother embodiment, a gene comprises the gene promoter. In anotherembodiment, a gene comprises its enhancer regions. In anotherembodiment, a gene comprises 5′ non-coding sequences. In anotherembodiment, a gene comprises 3′ non-coding sequences.

In one embodiment, the skilled artisan would appreciate that DNAcomprises a gene, which may include upstream and downstream sequences,as well as the coding region of the gene. In another embodiment, DNAcomprises a cDNA (complementary DNA). One of ordinary skill in the artwould appreciate that cDNA may encompass synthetic DNA reversetranscribed from RNA through the action of a reverse transcriptase. ThecDNA may be single stranded or double stranded and can include strandsthat have either or both of a sequence that is substantially identicalto a part of the RNA sequence or a complement to a part of the RNAsequence. Further, cDNA may include upstream and downstream regulatorysequences. In still another embodiment, DNA comprises CDS (completecoding sequence). One of ordinary skill in the art would appreciate thatCDS may encompass a DNA sequence, which encodes a full-length protein orpolypeptide. A CDS typically begins with a start codon (“ATG”) and endsat (or one before) the first in-frame stop codon (“TAA”, “TAG”, or“TGA”). The skilled artisan would recognize that a cDNA, in oneembodiment, comprises a CDS.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “isolated polynucleotide” are used interchangeablyherein. These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA or hybrid thereof, that issingle- or double-stranded, linear or branched, and that optionallycontains synthetic, non-natural or altered nucleotide bases. The termsalso encompass RNA/DNA hybrids.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression mediated by small double stranded RNAs. Itis the process of sequence-specific, post-transcriptional gene silencingin animals and plants, initiated by inhibitory RNA (iRNA) that ishomologous in its duplex region to the sequence of the silenced gene.The gene may be endogenous or exogenous to the organism, presentintegrated into a chromosome or present in a transfection vector that isnot integrated into the genome. The expression of the gene is eithercompletely or partially inhibited. RNAi may also be considered toinhibit the function of a target RNA; the function of the target RNA maybe complete or partial.

Typically, the term RNAi molecule refers to single- or double-strandedRNA molecules comprising both a sense and antisense sequence. Forexample, the RNA interference molecule can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprisescomplementarity to a target nucleic acid molecule. Alternatively theRNAi molecule can be a single-stranded hairpin polynucleotide havingself-complementary sense and antisense regions, wherein the antisenseregion comprises complementarity to a target nucleic acid molecule or itcan be a circular single-stranded. polynucleotide having two or moreloop structures and a stem comprising self-complementary sense andantisense regions, wherein the antisense region comprisescomplementarity to a target nucleic acid molecule, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active molecule capable of mediating RNAi.

The terms “complementary” or “complement thereof” are used herein torefer to the sequences of polynucleotides which is capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. This term isapplied to pairs of polynucleotides based solely upon their sequencesand not any particular set of conditions under which the twopolynucleotides would actually bind.

The term “construct” as used herein refers to an artificially assembledor isolated nucleic acid molecule which includes the polynucleotide ofinterest. In general, a construct may include the polynucleotide orpolynucleotides of interest, a marker gene which in some cases can alsobe a gene of interest and appropriate regulatory sequences. It should beappreciated that the inclusion of regulatory sequences in a construct isoptional, for example, such sequences may not be required in situationswhere the regulatory sequences of a host cell are to be used. The termconstruct includes vectors but should not be seen as being limitedthereto.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation.

The terms “promoter element,” “promoter,” or “promoter sequence” as usedherein, refer to a DNA sequence that is located at the 5′ end (i.e.precedes) the coding region of a DNA polymer. The location of mostpromoters known in nature precedes the transcribed region. The promoterfunctions as a switch, activating the expression of a gene. If the geneis activated, it is said to be transcribed, or participating intranscription. Transcription involves the synthesis of mRNA from thegene. The promoter, therefore, serves as a transcriptional regulatoryelement and also provides a site for initiation of transcription of thegene into mRNA.

As used herein, the term an “enhancer” refers to a DNA sequence whichcan stimulate promoter activity and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue-specificity of a promoter.

The term “expression”, as used herein, refers to the production of afunctional end-product e.g., an mRNA or a protein.

The term “gene edited plant” refers to a plant comprising at least onecell comprising at least one gene edited by man. The gene editingincludes deletion, insertion, silencing, or repression, such as of the“native genome” of the cell. Methods for creating a gene edited plantinclude techniques such as zinc-finger nucleases (ZFN), transcriptionactivator-like effector nucleases (TALEN), and clustered regularlyinterspersed short palindromic repeats (CRISPR)/Cas systems.

The term “genetically modified plant” refers to a plant comprising atleast one cell genetically modified by man. The genetic modificationincludes modification of an endogenous gene(s), for example byintroducing mutation(s) deletions, insertions, transposable element(s)and the like into an endogenous polynucleotide or gene of interest.Additionally, or alternatively, the genetic modification includestransforming the plant cell with heterologous polynucleotide. A“genetically modified plant” and a “corresponding unmodified plant” asused herein refer to a plant comprising at least one geneticallymodified cell and to a plant of the same type lacking said modification,respectively.

One of ordinary skill in the art would appreciate that a geneticallymodified plant may encompass a plant comprising at least one cellgenetically modified by man. In some embodiments, the geneticmodification includes modification of an endogenous gene(s), for exampleby introducing mutation(s) deletions, insertions, transposableelement(s) and the like into an endogenous polynucleotide or gene ofinterest. Additionally, or alternatively, in some embodiments, thegenetic modification includes transforming at least one plant cell witha heterologous polynucleotide or multiple heterologous polynucleotides.The skilled artisan would appreciate that a genetically modified plantcomprising transforming at least one plant cell with a heterologouspolynucleotide or multiple heterologous polynucleotides may in certainembodiments be termed a “transgenic plant”.

A skilled artisan would appreciate that a comparison of a “geneticallymodified plant” to a “corresponding unmodified plant” as used hereinencompasses comparing a plant comprising at least one geneticallymodified cell and to a plant of the same type lacking the modification.

The skilled artisan would appreciate that the term “transgenic” whenused in reference to a plant as disclosed herein encompasses a plantthat contains at least one heterologous transcribable polynucleotide inone or more of its cells. The term “transgenic material” encompassesbroadly a plant or a part thereof, including at least one cell, multiplecells or tissues that contain at least one heterologous polynucleotidein at least one of cell. Thus, comparison of a “transgenic plant” and a“corresponding non transgenic plant”, or of a “genetically modifiedplant comprising at least one cell having altered expression, whereinsaid plant comprising at least one cell comprising a heterologoustranscribable polynucleotide” and a “corresponding unmodified plant”encompasses comparison of the “transgenic plant” or “geneticallymodified plant” to a plant of the same type lacking said heterologoustranscribable polynucleotide. A skilled artisan would appreciate that,in some embodiments, a “transcribable polynucleotide” comprises apolynucleotide that can be transcribed into an RNA molecule by an RNApolymerase.

The terms “transformants” or “transformed cells” include the primarytransformed cell and cultures derived from that cell without regard tothe number of transfers. All progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same functionality as screened for in the originallytransformed cell are included in the definition of transformants.

Transformation of a cell may be stable or transient. The term “transienttransformation” or “transiently transformed” refers to the introductionof one or more exogenous polynucleotides into a cell in the absence ofintegration of the exogenous polynucleotide into the host cell's genome.In contrast, the term “stable transformation” or “stably transformed”refers to the introduction and integration of one or more exogenouspolynucleotides into the genome of a cell. The term “stabletransformant” refers to a cell which has stably integrated one or moreexogenous polynucleotides into the genomic or organellar DNA. It is tobe understood that an organism or its cell transformed with the nucleicacids, constructs and/or vectors of the present invention can betransiently as well as stably transformed.

The skilled artisan would appreciate that the term “construct” mayencompass an artificially assembled or isolated nucleic acid moleculewhich includes the polynucleotide of interest. In general, a constructmay include the polynucleotide or polynucleotides of interest, a markergene which in some cases can also be a gene of interest and appropriateregulatory sequences. It should be appreciated that the inclusion ofregulatory sequences in a construct is optional, for example, suchsequences may not be required in situations where the regulatorysequences of a host cell are to be used. The term construct includesvectors but should not be seen as being limited thereto.

The skilled artisan would appreciate that the term “expression” mayencompass the production of a functional end-product e.g., an mRNA or aprotein.

Based on the co-expressed gene array disclosed in the present invention,a pathway from cholesterol to α-tomatine is proposed (FIG. 1). It hasbeen previously described that cholesterol is hydroxylated at C22 byGAME7 (US 2012/0159676) followed by GAME8 hydroxylation at the C26position. The 22,26-dihydroxycholesterol is than hydroxylated at C16 andoxidized at C22 followed by closure of the E-ring by GAME11 and GAME6 toform the furostanol-type aglycone. This order of reactions is supportedby the finding of the present invention showing the accumulation ofcholestanol-type saponins, lacking hydroxylation at C16 and thehemi-acetal E-ring when silencing GAME11 (FIGS. 8A-D). Thefurostanol-intermediate is oxidized by GAME4 to its 26-aldehyde which isthe substrate for transamination catalyzed by GAME12. Nucleophilicattack of the amino-nitrogen at C22 leads to the formation oftomatidenol which is dehydrogenated to tomatidine. Tomatidine issubsequently converted by GAME1 to T-Gal (Itkin et al., 2011 supra).T-Gal in its turn is glucosylated by GAME17 into γ-tomatine, which isfurther glucosylated by GAME18 to β1-tomatine that is finally convertedto α-tomatine by GAME2 (FIG. 1).

The present invention now shows that by modifying expression of anenzyme and/or other protein involved in the biosynthetic pathway, thelevel of steroidal alkaloids, steroidal glycoalkaloids and optionallysteroidal saponin can be altered.

Silencing of a single gene co-expressed with the clusteredenzyme-encoding gene in potato plant, resulted in significant reductionin the amount of the steroidal glycoalkaloids α-chaconine andα-solanine, while overexpression of this gene resulted in significantincrease in the content of these substances (FIGS. 5A-5D and 6). Thisgene was found to include coding sequence comprising an AP2 domain, andtherefore postulated to be a transcription factor, designated hereinGAME9-transcription factor, encoded by GAME9.

A genetically modified or gene edited plant comprising at least one cellhaving altered expression of at least one gene selected from the groupconsisting of a gene encoding GAME9-transcription factor, a geneencoding 2-oxoglutarate-dependent dioxygenase, a gene encoding basichelix-loop-helix (BHLH)-transcription factor or a combination thereof,wherein the genetically modified or gene edited plant has an alteredcontent of at least one steroidal alkaloid or a glycosylated derivativethereof compared to a corresponding unmodified or unedited plant, hasbeen produced. As exemplified herein for 2-oxoglutarate-dependentdioxygenase (GAME11), manipulating the expression of the genes of thepresent invention can further lead to the manipulation of steroidalsaponin synthesis.

Thus, according to additional aspect, the present invention provides agenetically modified or gene edited organism comprising at least onecell having altered expression of at least one gene selected from thegroup consisting of a gene encoding GAME9-transcription factor, a geneencoding 2-oxoglutarate-dependent dioxygenase, a gene encoding basichelix-loop-helix (BHLH)-transcription factor or a combination thereofcompared to an unmodified or unedited organism, wherein the geneticallymodified or gene edited organism has an altered content of at least onecompound selected from steroidal saponin, steroidal alkaloid andglycosylated derivatives thereof compared to a corresponding unmodifiedor unedited organism.

Unexpectedly, the present invention now shows that SGA levels can beseverely reduced in potato tubers by modifying expression of an enzymeand/or transcription factors involved in the steroidal alkaloidsbiosynthetic pathway.

According to certain embodiments, the expression of the at least onegene selected from the group consisting of a gene encodingGAME9-transcription factor, a gene encoding 2-oxoglutarate-dependentdioxygenase, a gene encoding BHLH-transcription factor or thecombination thereof in the genetically modified or gene edited plant isinhibited compared to its expression in the corresponding unmodified orunedited plant, thereby the genetically modified or gene edited plantcomprises reduced content of at least one steroidal alkaloid or aglycosylated derivative thereof compared to a corresponding unmodifiedor unedited plant.

According to certain embodiments, the genetically modified or geneedited plant comprises non-toxic amount of steroidal alkaloid or aglycosylated derivative thereof. As used herein, the term “non-toxicamount” refers to less than 200 mg of antinutritional steroidal;alkaloids or glycoalkaloids per kilogram fresh weight of an edible plantpart. According to certain exemplary embodiments, the geneticallymodified or gene edited plant comprises non-detectable amount ofantinutritional steroidal alkaloid or a glycosylated derivative thereof.

Down-regulation or inhibition of the gene expression can be effected onthe genomic and/or the transcript level using a variety of moleculesthat interfere with transcription and/or translation (e.g., antisense,siRNA, Ribozyme, or DNAzyme), or on the protein level using, e.g.,antagonists, enzymes that cleave the polypeptide, and the like.

According to certain exemplary embodiments, the genetically modified orgene edited plant is a transgenic plant comprising at least one cellcomprising at least one silencing molecule targeted to a gene selectedfrom the group consisting of GAME9, GAME11, BHLH, or GAME15. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, the organism comprising the silencingmolecule has an elevated content of at least one steroidal saponin or aderivative thereof compared to a corresponding non-transgenic plant.

The silencing molecule target to at least one of GAME9, GAME11 and BHLHcan be designed as is known to a person skilled in the art. According tocertain embodiments, the silencing molecule comprises a polynucleotidehaving a nucleic acid sequence substantially complementary to a regionof the GAME9 gene, the gene having the nucleic acids sequence set forthin any one of SEQ ID NO:4 and SEQ ID NO:6.

According to certain additional embodiments, the silencing moleculecomprises a polynucleotide having a nucleic acid sequence substantiallycomplementary to a region of the GAME11 gene, the gene having thenucleic acids sequence set forth in any one of SEQ ID NO:10 and SEQ IDNO:12.

According to certain further embodiments, the silencing moleculecomprises a polynucleotide having a nucleic acid sequence substantiallycomplementary to a region of the BHLH gene, the gene having the nucleicacids sequence set forth in any one of SEQ ID NO:15 and SEQ ID NO:17.

According to certain additional embodiments, the silencing moleculecomprises a polynucleotide having a nucleic acid sequence substantiallycomplementary to a region of the GAME15 gene, the gene having thenucleic acids sequence set forth in any one of SEQ ID NO:44, SEQ IDNO:45, and SEQ ID NO:46.

Antisense Molecules

Antisense technology is the process in which an antisense RNA or DNAmolecule interacts with a target sense DNA or RNA strand. A sense strandis a 5′ to 3′ mRNA molecule or DNA molecule. The complementary strand,or mirror strand, to the sense is called an antisense. When an antisensestrand interacts with a sense mRNA strand, the double helix isrecognized as foreign to the cell and will be degraded, resulting inreduced or absent protein production. Although DNA is already a doublestranded molecule, antisense technology can be applied to it, building atriplex formation.

One skilled in the art would appreciate that the terms “complementary”or “complement thereof” are used herein to encompass the sequences ofpolynucleotides which is capable of forming Watson & Crick base pairingwith another specified polynucleotide throughout the entirety of thecomplementary region. This term is applied to pairs of polynucleotidesbased solely upon their sequences and not any particular set ofconditions under which the two polynucleotides would actually bind.

RNA antisense strands can be either catalytic or non-catalytic. Thecatalytic antisense strands, also called ribozymes, cleave the RNAmolecule at specific sequences. A non-catalytic RNA antisense strandblocks further RNA processing.

Antisense modulation of cells and/or tissue levels of the GAME9, GAME11,and BHLH gene or any combination thereof may be effected by transformingthe organism cells or tissues with at least one antisense compound,including antisense DNA, antisense RNA, a ribozyme, DNAzyme, a lockednucleic acid (LNA) and an aptamer. In some embodiments the molecules arechemically modified. In other embodiments the antisense molecule isantisense DNA or an antisense DNA analog.

Antisense modulation of cells and/or tissue levels of the GAME15 gene orany combination thereof may be effected by transforming the organismcells or tissues with at least one antisense compound, includingantisense DNA, antisense RNA, a ribozyme, DNAzyme, a locked nucleic acid(LNA), and an aptamer. In some embodiments, the molecules are chemicallymodified. In other embodiments, the antisense molecule is antisense DNAor an antisense DNA analog.

RNA Interference (RNAi) Molecules

RNAi refers to the introduction of homologous double stranded RNA(dsRNA) to target a specific gene product, resulting in posttranscriptional silencing of that gene. This phenomenon was firstreported in Caenorhabditis elegans by Guo and Kemphues (1995, Cell,81(4):611-620) and subsequently Fire et al. (1998, Nature 391:806-811)discovered that it is the presence of dsRNA, formed from the annealingof sense and antisense strands present in the in vitro RNA preps, thatis responsible for producing the interfering activity

In both plants and animals, RNAi is mediated by RNA-induced silencingcomplex (RISC), a sequence-specific, multicomponent nuclease thatdestroys messenger RNAs homologous to the silencing trigger. RISC isknown to contain short RNAs (approximately 22 nucleotides) derived fromthe double-stranded RNA trigger. The short-nucleotide RNA sequences arehomologous to the target gene that is being suppressed. Thus, theshort-nucleotide sequences appear to serve as guide sequences toinstruct a multicomponent nuclease, RISC, to destroy the specific mRNAs.

The dsRNA used to initiate RNAi, may be isolated from native source orproduced by known means, e.g., transcribed from DNA. Plasmids andvectors for generating RNAi molecules against target sequence are nowreadily available from commercial sources.

The dsRNA can be transcribed from the vectors as two separate strands.In other embodiments, the two strands of DNA used to form the dsRNA maybelong to the same or two different duplexes in which they each formwith a DNA strand of at least partially complementary sequence. When thedsRNA is thus-produced, the DNA sequence to be transcribed is flanked bytwo promoters, one controlling the transcription of one of the strands,and the other that of the complementary strand. These two promoters maybe identical or different. Alternatively, a single promoter can derivethe transcription of single-stranded hairpin polynucleotide havingself-complementary sense and antisense regions that anneal to producethe dsRNA.

One skilled in the art would appreciate that the terms “promoterelement,” “promoter,” or “promoter sequence” may encompass a DNAsequence that is located at the 5′ end (i.e. precedes) the coding regionof a DNA polymer. The location of most promoters known in natureprecedes the transcribed region. The promoter functions as a switch,activating the expression of a gene. If the gene is activated, it issaid to be transcribed, or participating in transcription. Transcriptioninvolves the synthesis of mRNA from the gene. The promoter, therefore,serves as a transcriptional regulatory element and also provides a sitefor initiation of transcription of the gene into mRNA.

Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. RNA molecules containing a nucleotide sequence identical toa portion of the target gene are preferred for inhibition. RNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Thus, sequence identity may be optimized by sequence comparison andalignment algorithms known in the art (see Gribskov and Devereux,Sequence Analysis Primer, Stockton Press, 1991, and references citedtherein) and calculating the percent difference between the nucleotidesequences by, for example, the Smith-Waterman algorithm as implementedin the BESTFIT software program using default parameters (e.g.,University of Wisconsin Genetic Computing Group). Greater than 90%sequence identity, or even 100% sequence identity, between theinhibitory RNA and the portion of the target gene is preferred.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript. The length of the identical nucleotidesequences may be at least 25, 50, 100, 200, 300 or 400 bases. There isno upper limit on the length of the dsRNA that can be used. For example,the dsRNA can range from about 21 base pairs (bp) of the gene to thefull length of the gene or more.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression mediated by small double stranded RNAs. Itis the process of sequence-specific, post-transcriptional gene silencingin animals and plants, initiated by inhibitory RNA (iRNA) that ishomologous in its duplex region to the sequence of the silenced gene.The gene may be endogenous or exogenous to the organism, presentintegrated into a chromosome or present in a transfection vector that isnot integrated into the genome. The expression of the gene is eithercompletely or partially inhibited. RNAi may also be considered toinhibit the function of a target RNA; the function of the target RNA maybe complete or partial.

One of ordinary skill in the art would appreciate that the term RNAimolecule refers to single- or double-stranded RNA molecules comprisingboth a sense and antisense sequence. For example, the RNA interferencemolecule can be a double-stranded polynucleotide molecule comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises complementarity to a target nucleic acid molecule.Alternatively the RNAi molecule can be a single-stranded hairpinpolynucleotide having self-complementary sense and antisense regions,wherein the antisense region comprises complementarity to a targetnucleic acid molecule or it can be a circular single-strandedpolynucleotide having two or more loop structures and a stern comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises complementarity to a target nucleic acid molecule, andwherein the circular polynucleotide can be processed either in vivo orin vitro to generate an active molecule capable of mediating RNAi.

The present invention contemplates the use of RNA interference (RNAi) todown regulate the expression of GAME9, GAME11, BHLH, or GAME15 or acombination thereof to attenuate the level of steroidalalkaloids/glycoalkaloids in plants. In both plants and animals, RNAi ismediated by RNA-induced silencing complex (RISC), a sequence-specific,multicomponent nuclease that destroys messenger RNAs homologous to thesilencing trigger. RISC is known to contain short RNAs (approximately 22nucleotides) derived from the double-stranded RNA trigger. Theshort-nucleotide RNA sequences are homologous to the target gene that isbeing suppressed. Thus, the short-nucleotide sequences appear to serveas guide sequences to instruct a multicomponent nuclease, RISC, todestroy the specific mRNAs.

The dsRNA used to initiate RNAi, may be isolated from native source orproduced by known means, e.g., transcribed from DNA Plasmids and vectorsfor generating RNAi molecules against target sequence are now readilyavailable as exemplified herein below.

The dsRNA can be transcribed from the vectors as two separate strands.In other embodiments, the two strands of DNA used to form the dsRNA maybelong to the same or two different duplexes in which they each formwith a DNA strand of at least partially complementary sequence. When thedsRNA is thus-produced, the DNA sequence to be transcribed is flanked bytwo promoters, one controlling the transcription of one of the strands,and the other that of the complementary strand. These two promoters maybe identical or different. Alternatively, a single promoter can derivethe transcription of single-stranded hairpin polynucleotide havingself-complementary sense and antisense regions that anneal to producethe dsRNA.

Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. RNA molecules containing a nucleotide sequence identical toa portion of the target gene are preferred for inhibition. RNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Thus, sequence identity may optimized by sequence comparison andalignment algorithms known in the art (see Gribskov and Devereux,Sequence Analysis Primer, Stockton Press, 1991, and references citedtherein) and calculating the percent difference between the nucleotidesequences by, for example, the Smith-Waterman algorithm as implementedin the BESTFIT software program using default parameters (e.g.,University of Wisconsin Genetic Computing Group). Greater than 90%sequence identity, or even 100% sequence identity, between theinhibitory RNA and the portion of the target gene is preferred.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript. The length of the identical nucleotidesequences may be at least 25, 50, 100, 200, 300 or 400 bases. There isno upper limit on the length of the dsRNA that can be used. For example,the dsRNA can range from about 21 base pairs (bp) of the gene to thefull length of the gene or more.

According to certain currently typical embodiments, the silencingmolecule is RNAi targeted to the GAME9 gene, comprising the nucleic acidsequence set forth in SEQ ID NO:18 or a complementary sequence thereof.According to certain currently typical embodiments, the silencingmolecule is RNAi targeted to the cellulose synthase like GAME15 gene,comprising the nucleic acid sequence set forth any one of in SEQ ID NOs:44 to 46 or a complementary sequence thereof.

According to additional typical embodiments, the silencing molecule isRNAi targeted to the GAME11 gene, comprising the nucleic acid sequenceset forth in SEQ ID NO:19 or a complementary sequence thereof.

According to additional typical embodiments, the silencing molecule isRNAi targeted to the GAME15 gene, comprising the nucleic acid sequenceset forth in SEQ ID NO:44 or a complementary sequence thereof; SEQ IDNO:45 or a complementary sequence thereof; and/or SEQ NO:46 or acomplementary sequence thereof.

Co-Suppression Molecules

Another agent capable of down-regulating the expression of GAME9 orGAME11, or a combination thereof is a Co-Suppression molecule.Co-suppression is a post-transcriptional mechanism where both thetransgene and the endogenous gene are silenced.

Another agent capable of down-regulating the expression of GAME15 is aCo-Suppression molecule. Co-suppression is a post-transcriptionalmechanism where both the transgene and the endogenous gene are silenced.

DNAzyme Molecules

Another agent capable of down-regulating the expression of GAME9,GAME11, BHLH, or GAME15 is a DNAzyme molecule, which is capable ofspecifically cleaving an mRNA transcript or a DNA sequence of the GAME9,GAME 11, BHLH, or GAME15. DNAzymes are single-stranded polynucleotidesthat are capable of cleaving both single- and double-stranded targetsequences. A general model (the “10-23” model) for the DNAzyme has beenproposed. “10-23” DNAzymes have a catalytic domain of 15deoxyribonucleotides, flanked by two substrate-recognition domains ofseven to nine deoxyribonucleotides each. This type of DNAzyme caneffectively cleave its substrate RNA at purine:pyrimidine junctions (forreview of DNAzymes, see: Khachigian, L. M. (2002) Curr Opin Mol Ther 4,119-121).

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single- and double-stranded target cleavage sitesare disclosed in U.S. Pat. No. 6,326,174.

Enzymatic Oligonucleotide

The terms “enzymatic nucleic acid molecule” or “enzymaticoligonucleotide” refers to a nucleic acid molecule which hascomplementarity in a substrate binding region to a specified genetarget, and also has an enzymatic activity which is active tospecifically cleave target RNA of GAME9, GAME11, BHLH, or GAME15,thereby silencing each of the genes. The complementary regions allowsufficient hybridization of the enzymatic nucleic acid molecule to thetarget RNA and subsequent cleavage. The term enzymatic nucleic acid isused interchangeably with for example, ribozymes, catalytic RNA,enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme,catalytic oligonucleotide, nucleozyme, DNAzyme, RNAenzyme. The specificenzymatic nucleic acid molecules described in the instant applicationare not limiting and an enzymatic nucleic acid molecule of thisinvention requires a specific substrate binding site which iscomplementary to one or more of the target nucleic acid regions, andthat it have nucleotide sequences within or surrounding that substratebinding site which impart a nucleic acid cleaving and/or ligationactivity to the molecule. U.S. Pat. No. 4,987,071 discloses examples ofsuch molecules.

Mutagenesis

Altering the expression of endogenous GAME9, GAME11, BHLH, or GAME15genes can be also achieved by the introduction of one or more pointmutations into a nucleic acid molecule encoding the correspondingproteins. Mutations can be introduced using, for example, site-directedmutagenesis (see, e.g. Wu Ed., 1993 Meth. In Enzymol. Vol 217, SanDiego: Academic Press; Higuchi, “Recombinant PCR” in Innis et al. Eds.,1990 PCR Protocols, San Diego: Academic Press, Inc). Such mutagenesiscan be used to introduce a specific, desired amino acid insertion,deletion or substitution. Several technologies for targeted mutagenesisare based on the targeted induction of double-strand breaks (DSBs) inthe genome followed by error-prone DNA repair. Mostly commonly used forgenome editing by these methods are custom designed nucleases, includingzinc finger nucleases and Xanthomonas-derived transcriptionactivator-like effector nuclease (TALEN) enzymes.

In some embodiments, when the expression of the at least one gene orcombination thereof is altered, said altering comprises mutagenizing theat least one gene, said mutation present within a coding region of saidat least one gene, or a regulatory sequence of said at least one gene,or a combination thereof.

Various types of mutagenesis can be used to modify GAME9, GAME11, BHLH,or GAME15 and their encoded polypeptides in order to produceconservative or non-conservative variants. Any available mutagenesisprocedure can be used. In some embodiments, the mutagenesis procedurecomprises site-directed point mutagenesis. In some embodiments, themutagenesis procedure comprises random point mutagenesis. In someembodiments, the mutagenesis procedure comprises in vitro or in vivohomologous recombination (DNA shuffling). In some embodiments, themutagenesis procedure comprises mutagenesis using uracil-containingtemplates. In some embodiments, the mutagenesis procedure comprisesoligonucleotide-directed mutagenesis. In some embodiments, themutagenesis procedure comprises phosphorothioate-modified DNAmutagenesis. In some embodiments, the mutagenesis procedure comprisesmutagenesis using gapped duplex DNA. In some embodiments, themutagenesis procedure comprises point mismatch repair. In someembodiments, the mutagenesis procedure comprises mutagenesis usingrepair-deficient host strains. In some embodiments, the mutagenesisprocedure comprises restriction-selection and restriction-purification.In some embodiments, the mutagenesis procedure comprises deletionmutagenesis. In some embodiments, the mutagenesis procedure comprisesmutagenesis by total gene synthesis. In some embodiments, themutagenesis procedure comprises double-strand break repair. In someembodiments, the mutagenesis procedure comprises mutagenesis by chimericconstructs. In some embodiments, the mutagenesis procedure comprisesmutagenesis by CRISPR/Cas. In some embodiments, the mutagenesisprocedure comprises mutagenesis by zinc-finger nucleases (ZFN). In someembodiments, the mutagenesis procedure comprises mutagenesis bytranscription activator-like effector nucleases (TALEN). In someembodiments, the mutagenesis procedure comprises any other mutagenesisprocedure known to a person skilled in the art.

In some embodiments, mutagenesis can be guided by known informationabout the naturally occurring molecule and/or the mutated molecule. Byway of example, this known information may include sequence, sequencecomparisons, physical properties, crystal structure and the like. Insome embodiments, the mutagenesis is essentially random. In someembodiments the mutagenesis procedure is DNA shuffling.

A skilled artisan would appreciate that clustered regularly interspacedshort palindromic repeats (CRISPR)/CRISPR associated protein (Cas)system comprises genome engineering tools based on the bacterialCRISPR/Cas prokaryotic adaptive immune system. This RNA-based technologyis very specific and allows targeted cleavage of genomic DNA guided by acustomizable small noncoding RNA, resulting in gene modifications byboth non-homologous end joining (NHEJ) and homology-directed repair(HDR) mechanisms (Belhaj K. et al., 2013. Plant Methods 2013, 9:39). Insome embodiments, a CRISPR/Cas system comprises a CRISPR/Cas9 system.

In some embodiments, a CRISPR/Cas system comprises a single-guide RNA(sgRNA) and/or a Cas protein known in the art. In some embodiments, aCRISPR/Cas system comprises a single-guide RNA (sgRNA) and/or a Casprotein newly created to cleave at a preselected site. The skilledartisan would appreciate that the terms “single-guide RNA”, “sgRNA”, and“gRNA” are interchangeable having all the same qualities and meanings,wherein an sgRNA may encompass a chimeric RNA molecule which is composedof a CRISPR RNA (crRNA) and trans-encoded CRISPR. RNA (tracrRNA). Insome embodiments, a crRNA is complementary to a preselected region ofGAME15 DNA, wherein the crRNA “targets” the CRISPR associatedpolypeptide (Cas) nuclease protein to the preselected target site.

In some embodiments, the length of crRNA sequence complementary is 19-22nucleotides long e.g., 19-22 consecutive nucleotides complementary tothe target site. In another embodiment, the length of crRNA sequencecomplementary to the region of DNA is about 15-30 nucleotides long. Inanother embodiment, the length of crRNA sequence complementary to theregion of DNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides long. In another embodiment, the length ofcrRNA sequence complementary to the region of DNA is 20 nucleotideslong. In some embodiments, the crRNA is located at the 5′ end of thesgRNA molecule. In another embodiment, the crRNA comprises 100%complementation within the preselected target sequence. In anotherembodiment, the crRNA comprises at least 80% complementation within thepreselected target sequence. In another embodiment, the crRNA comprisesat least 85% complementation within the preselected target sequence. Inanother embodiment, the crRNA comprises at least 90% complementationwithin the preselected target sequence. In another embodiment, the crRNAcomprises at least 95% complementation within the preselected targetsequence. In another embodiment, the crRNA comprises at least 97%complementation within the preselected target sequence. In anotherembodiment, the crRNA comprises at least 99% complementation within thepreselected target sequence. In another embodiment, a tracrRNA is100-300 nucleotides long and provides a binding site for the Casnuclease, e.g., a Cas9 protein forming the CRISPR/Cas9 complex.

In one embodiment, a mutagenesis system comprises a CRISPR/Cas system.In another embodiment, a CRISPR/Cas system comprises a Cas nuclease anda gRNA molecule, wherein said gRNA molecule binds within saidpreselected endogenous target site thereby guiding said Cas nuclease tocleave the DNA within said preselected endogenous target site.

In some embodiments, a CRISPR/Cas system comprise an enzyme systemincluding a guide RNA sequence (“gRNA” or “sgRNA”) that contains anucleotide sequence complementary or substantially complementary to aregion of a target polynucleotide, for example a preselected endogenoustarget site, and a protein with nuclease activity.

In another embodiment, a CRISPR/Cas system comprises a Type I CRISPR-Cassystem, or a Type II CRISPR-Cas system, or a Type III CRISPR-Cas system,or derivatives thereof. In another embodiment, a CRISPR-Cas systemcomprises an engineered and/or programmed nuclease system derived fromnaturally accruing CRISPR-Cas systems. In another embodiment, aCRISPR-Cas system comprises engineered and/or mutated Cas proteins. Inanother embodiment, a CRISPR-Cas system comprises engineered and/orprogrammed guide RNA.

A skilled artisan would appreciate that a guide RNA may containnucleotide sequences other than the region complementary orsubstantially complementary to a region of a target DNA sequence, forexample a preselected endogenous target site. In another embodiment, aguide RNA comprises a crRNA or a derivative thereof. In anotherembodiment, a guide RNA comprises a crRNA: tracrRNA chimera.

In another embodiment, a gRNA molecule comprises a domain that iscomplementary to and binds to a preselected endogenous target site on atleast one homologous chromosome. In another embodiment, a gRNA moleculecomprises a domain that is complementary to and binds to a polymorphicallele on at least one homologous chromosome. In another embodiment, agRNA molecule comprises a domain that is complementary to and binds to apreselected endogenous target site on both homologous chromosomes. Inanother embodiment, a gRNA molecule comprises a domain that iscomplementary to and binds to polymorphic alleles on both homologouschromosomes.

Cas enzymes comprise RNA-guided DNA endonuclease able to makedouble-stranded breaks (DSB) in DNA. The term “Cas enzyme” may be usedinterchangeably with the terms “CRISPR-associated endonucleases” or“CRISPR-associated polypeptides” having all the same qualities andmeanings. In one embodiment, a Cas enzyme is selected from the groupcomprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9,Cas10, C2c1, CasX, NgAgo, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1,Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5,Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1,Csx15, Csf1, Csf2, Csf3, and Csf4, or homologs thereof, or modifiedversions thereof. In another embodiment, a Cas enzyme comprises Cas9. Inanother embodiment, a Cas enzyme comprises Cas1. In another embodiment,a Cas enzyme comprises Cas1B. In another embodiment, a Cas enzymecomprises Cas2. In another embodiment, a Cas enzyme comprises Cas3. Inanother embodiment, a Cas enzyme comprises Cas4. In another embodiment,a Cas enzyme comprises Cas5. In another embodiment, a Cas enzymecomprises Cas6. In another embodiment, a Cas enzyme comprises Cas7. Inanother embodiment, a Cas enzyme comprises Cas8. In another embodiment,a Cas enzyme comprises Cas10. In another embodiment, a Cas enzymecomprises Cpf1. In another embodiment, a Cas enzyme comprises Csy1. Inanother embodiment, a Cas enzyme comprises Csy2. In another embodiment,a Cas enzyme comprises Csy3. In another embodiment, a Cas enzymecomprises Cse1 another embodiment, a Cas enzyme comprises Cse2. Inanother embodiment, a Cas enzyme comprises Csc1. In another embodiment,a Cas enzyme comprises Csc2. In another embodiment, a Cas enzymecomprises Csa5. In another embodiment, a Cas enzyme comprises Csn2. Inanother embodiment, a Cas enzyme comprises Csm2. In another embodiment,a Cas enzyme comprises Csm3. In another embodiment, a Cas enzymecomprises Csm4. In another embodiment, a Cas enzyme comprises Csm5. Inanother embodiment, a Cas enzyme comprises Csm6. In another embodiment,a Cas enzyme comprises Cmr1. In another embodiment, a Cas enzymecomprises Cmr3. In another embodiment, a Cas enzyme comprises Cmr4. Inanother embodiment, a Cas enzyme comprises Cmr5. In another embodiment,a Cas enzyme comprises Cmr6. In another embodiment, a Cas enzymecomprises Csb1. In another embodiment, a Cas enzyme comprises Csb2, Inanother embodiment, a Cas enzyme comprises Csb3. In another embodiment,a Cas enzyme comprises Csx17. In another embodiment, a Cas enzymecomprises Csx14. In another embodiment, a Cas enzyme comprises Csx10. Inanother embodiment, a Cas enzyme comprises Csx16, CsaX. In anotherembodiment, a Cas enzyme comprises Csx3, In another embodiment, a Casenzyme comprises Csx1, Csx15, Csf1. In another embodiment, a Cas enzymecomprises Csf2. In another embodiment, a Cas enzyme comprises Csf3. Inanother embodiment, a Cas enzyme comprises Csf4. In another embodiment,a Cas enzyme comprises Cpf1. In another embodiment, a Cas enzymecomprises C2c1. In another embodiment, a Cas enzyme comprises CasX. Inanother embodiment, a Cas enzyme comprises NgAgo. In another embodiment,a Cas enzyme is Cas homologue. In another embodiment, a Cas enzyme is aCas orthologue. In another embodiment, a Cas enzyme is a modified Casenzyme. In another embodiment, a Cas enzyme is any CRISPR-associatedendonucleases known in the art.

A skilled artisan would appreciate that the terms “zinc finger nuclease”or “ZFN” are interchangeable having all the same meanings and qualities,wherein a ZFN encompasses a chimeric protein molecule comprising atleast one zinc finger DNA binding domain operatively linked to at leastone nuclease capable of double-strand cleaving of DNA. In someembodiments, a ZFN system comprises a ZFN known in the art. In someembodiments, a ZFN system comprises a ZFN newly created to cleave apreselected site.

In some embodiments, a ZFN creates a double-stranded break at apreselected endogenous target site. In some embodiments, a ZFN comprisesa DNA-binding domain and a DNA-cleavage domain, wherein the DNA bindingdomain is comprised of at least one zinc finger and is operativelylinked to a DNA-cleavage domain. In another embodiment, a zinc fingerDNA-binding domain is at the N-terminus of the chimeric protein moleculeand the DNA-cleavage domain is located at the C-terminus of themolecule. In another embodiment, a zinc finger DNA-binding domain is atthe C-terminus of the chimeric protein molecule and the DNA-cleavagedomain is located at the N-terminus of the molecule. In anotherembodiment, a zinc finger binding domain encompasses the region in azinc finger nuclease that is capable of binding to a target locus, forexample a preselected endogenous target site as disclosed herein. Inanother embodiment, a zinc finger DNA-binding domain comprises a proteindomain that binds to a preselected endogenous target site on at leastone homologous chromosome. In another embodiment, a zinc fingerDNA-binding domain comprises a protein domain that binds to apolymorphic allele on at least one homologous chromosome. In anotherembodiment, a zinc finger DNA-binding domain comprises a protein domainthat binds to a preselected endogenous target site on both homologouschromosomes. In another embodiment, a zinc finger DNA-binding domaincomprises a protein domain that binds to polymorphic alleles on bothhomologous chromosomes.

The skilled artisan would appreciate that the term “chimeric protein” isused to describe a protein that has been expressed from a DNA moleculethat has been created by operatively joining two or more DNA fragments.The DNA fragments may be from the same species, or they may be from adifferent species. The DNA fragments may be from the same or a differentgene. The skilled artisan would appreciate that the term “DNA cleavagedomain” of a ZFN encompasses the region in the zinc finger nuclease thatis capable of breaking down the chemical bonds between nucleic acids ina nucleotide chain. Examples of proteins containing cleavage domainsinclude restriction enzymes, topoisomerases, recombinases, integrasesand DNAses.

In some embodiments, a TALEN system comprises a TAL effector DNA bindingdomain and a DNA cleavage domain, wherein said TAL effector DNA bindingdomain binds within said preselected endogenous target site, therebytargeting the DNA cleavage domain to cleave the DNA within saidpreselected endogenous target site.

A skilled artisan would appreciate that the terms “transcriptionactivator-like effector nuclease”, “TALEN”, and “TAL effector nuclease”may be used interchangeably having all the same meanings and qualities,wherein a TALEN encompasses a nuclease capable of recognizing andcleaving its target site, for example a preselected endogenous targetsite as disclosed herein. In another embodiment, a TALEN comprises afusion protein comprising a TALE domain and a nucleotide cleavagedomain. In another embodiment, a TALE domain comprises a protein domainthat binds to a nucleotide in a sequence-specific manner through one ormore TALE-repeat modules. A skilled artisan would recognize thatTALE-repeat modules comprise a variable number of about 34 amino acidrepeats that recognize plant DNA sequences. Further, repeat modules canbe rearranged according to a simple cipher to target new DNA sequences.In another embodiment, a TALE domain comprises a protein domain thatbinds to a preselected endogenous target site on at least one homologouschromosome. In another embodiment, a TALE domain comprises a proteindomain that binds to a polymorphic allele on at least one homologouschromosome. In another embodiment, a TALE domain comprises a proteindomain that binds to a preselected endogenous target site on bothhomologous chromosomes. In another embodiment, a TALE domain comprises aprotein domain that binds to polymorphic alleles on both homologouschromosomes.

In one embodiment, a TALE domain comprises at least one of theTALE-repeat modules. In another embodiment, a TALE domain comprises fromone to thirty TALE-repeat modules. In another embodiment, a TALE domaincomprises more than thirty repeat modules. In another embodiment, aTALEN fusion protein comprises an N-terminal domain, one or more ofTALE-repeat modules followed by a half-repeat module, a linker, and anucleotide cleavage domain.

Chemical mutagenesis using an agent such as Ethyl Methyl Sulfonate (EMS)can be employed to obtain a population of point mutations and screen formutants of the GAME9, GAME11, BHLH, or GAME15 genes that may becomesilent or down-regulated. In plants, methods relaying on introgressionof genes from natural populations can be used. Cultured and wild typesspecies are crossed repetitively such that a plant comprising a givensegment of the wild genome is isolated. Certain plant species, forexample, maize (corn) and snapdragon, have natural transposons. Thesetransposons are either autonomous, i.e. the transposase is locatedwithin the transposon sequence or non-autonomous, without a transposase.A skilled person can cause transposons to “jump” and create mutations.Alternatively, a nucleic acid sequence can be synthesized having randomnucleotides at one or more predetermined positions to generate randomamino acid substituting.

In some embodiments, the expression of endogenous GAME9, GAME11, BHLH,or GAME15 genes can be altered by the introduction of one or more pointmutations into their regulatory sequences. In some embodiments, theexpression of exogenous GAME9, GAME11, BHLH, or GAME15 genes can bealtered by the introduction of one or more point mutations into theirregulatory sequences. A skilled artisan would appreciate that“regulatory sequences” refers to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. In some embodiments, regulatory sequences comprise promoters.In some embodiments, regulatory sequences comprise translation leadersequences. In some embodiments, regulatory sequences comprise introns.In some embodiments, regulatory sequences comprise polyadenylationrecognition sequences. In some embodiments, regulatory sequencescomprise RNA processing sites. In some embodiments, regulatory sequencescomprise effector binding sites. In some embodiments, regulatorysequences comprise stem-loop structures.

A skilled artisan would appreciate that “promoter” refers to a DNAsequence capable of controlling the expression of a coding sequence orfunctional RNA. In some embodiments, a coding sequence is located 3′ toa promoter sequence. It is understood by those skilled in the art thatdifferent promoters may direct the expression of a gene in differenttissues or cell types, or at different stages of development, or inresponse to different environmental or physiological conditions. In someembodiments, the promoter comprises a constitutive promoter, i.e., apromoter that causes a gene to be expressed in most cell types at mosttimes. In some embodiments, the promoter comprises a regulated promoter,i.e., a promoter that causes a gene to be expressed in response tosporadic specific stimuli. It is further recognized that in many casesthe exact boundaries of regulatory sequences have not been completelydefined yet.

A skilled artisan would appreciate that the term “3′ non-codingsequences” or “transcription terminator” refers to DNA sequences locateddownstream of a coding sequence. In some embodiments, 3′ non-codingsequences comprise polyadenylation recognition sequences. In someembodiments, 3′ non-coding sequences comprise sequences encodingregulatory signals capable of affecting mRNA processing. In someembodiments, 3′ non-coding sequences comprise sequences encodingregulatory signals capable of affecting gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. In some embodiments, mutations in the 3′ non-coding sequencesaffect gene transcription. In some embodiments, mutations in the 3′non-coding sequences affect RNA processing. In some embodiments,mutations in the 3′ non-coding sequences affect gene stability. In someembodiments, mutations in the 3′ non-coding sequences affect translationof the associated coding sequence.

Biological Activity

In some embodiments, the biological activity of GAME9, GAME11, BHLH,GAME15 is altered compared with a control GAME9 enzyme, a control GAME11enzyme, a control BHLH enzyme, or a control GAME15 protein.

A skilled artisan would recognize that the term “biological activity”refers to any activity associated with a protein that can be measured byan assay. In some embodiments, the biological activity of GAME15comprises biosynthesis of steroidal alkaloids and glycosylatedderivatives thereof. In some embodiments, the biological activity ofGAME15 affect the levels of steroidal alkaloids in at least a part of aplant. In some embodiments, an altered biological activity comprisesincreased enzyme activity. In some embodiments, an altered biologicalactivity comprises decreased enzyme activity. In some embodiments, analtered biological activity comprises increased stability of thepolypeptide. In some embodiments, an altered biological activitycomprises decreased stability of the polypeptide.

In some embodiments, the altered biological activity comprises

-   -   increased enzyme activity of said cellulose synthase like gene        enzyme (GAME15); or    -   increased stability of said cellulose synthase like gene enzyme        (GAME15); or    -   decreased enzyme activity of said cellulose synthase like gene        enzyme (GAME15); or    -   decreased stability of said cellulose synthase like gene enzyme        (GAME15);        compared to the biological activity in an unmodified or unedited        plant.

In some embodiments, the biological activity of a GAME15 enzyme isincreased compared with a control GAME15 enzyme. In some embodiments,the biological activity of a GAME15 enzyme is decreased compared with acontrol GAME15 enzyme. In some embodiments, a GAME15 enzyme hasincreased stability compared with a control GAME15 enzyme. In someembodiments, a GAME15 enzyme has decreased stability compared with acontrol GAME15 enzyme.

Overexpression

According to yet additional embodiments the present invention provides agenetically modified or gene edited plant having enhanced expression ofat least one gene selected from the group consisting of a gene encodingGAME9-transcription factor, a gene encoding 2-oxoglutarate-dependentdioxygenase, a gene encoding basic helix-loop-helix transcription factor(BHLH), a gene encoding GAME15, or a combination thereof, wherein thegenetically modified or gene edited plant has an increased amount of atleast one steroidal alkaloid or a glycosylated derivative thereofcompared to a corresponding unmodified or unedited plant. In plants,steroidal alkaloids play a role in protecting the plant from variouspathogens. Steroidal alkaloids are referred to as phytoanticipins, i.e.low molecular weight anti-microbial compounds that are present in theplant before challenge by microorganisms or produced after infectionsolely from preexisting constituents. Over-expression of GAME9, GAME11,BHLH, GAME15, or any combination thereof in non-edible parts of theplant can thus enhance the plant resistance tosteroidal-alkaloid-sensitive pathogens.

Transgenic Plants

Cloning of a polynucleotide encoding a protein of the present inventionselected from the group consisting of GAME9-transcription factor,2-oxoglutarate-dependent dioxygenase, BHLH transcription factor, GAME15or a molecule that silences a gene encoding same can be performed by anymethod as is known to a person skilled in the art. Cloning of apolynucleotide encoding a GAME15 protein of the present invention or amolecule that silences a gene encoding same can be performed by anymethod as is known to a person skilled in the art. Various DNAconstructs may be used to express the desired gene or silencing moleculetargeted to the gene in a desired organism.

According to certain embodiments, the gene or a silencing moleculetargeted thereto form part of an expression vector comprising allnecessary elements for expression of the gene or its silencing molecule.According to certain embodiments, the expression is controlled by aconstitutive promoter. According to certain embodiments, theconstitutive promoter is specific to a plant tissue. According to theseembodiments, the tissue specific promoter is selected from the groupconsisting of root, tuber, leaves and fruit specific promoter. Rootspecific promoters are described, e.g. in Martinez, E. et al, 2003.Curr. Biol. 13:1435-1441. Fruit specific promoters are described amongothers in Estornell L. H et al. 2009. Plant Biotechnol. J. 7:298-309 andFernandez A. I. Et al. 2009 Plant Physiol. 151:1729-1740. Tuber specificpromoters are described, e.g. in Rocha-Sosa M, et al., 1989. EMBO J.8:23-29; McKibbin R. S. et al., 2006. Plant Biotechnol J. 4(4409-18.Leaf specific promoters are described, e.g. in Yutao Yang, Guodong Yang,Shijuan Liu, Xingqi Guo and Chengchao Zheng. Science in China Series C:Life Sciences. 46: 651-660.

According to certain embodiments, the expression vector furthercomprises regulatory elements at the 3′ non-coding sequence. As usedherein, the “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht I L et al. (1989.Plant Cell 1:671-680).

Those skilled in the art will appreciate that the various components ofthe nucleic acid sequences and the transformation vectors described inthe present invention are operatively linked, so as to result inexpression of said nucleic acid or nucleic acid fragment. Techniques foroperatively linking the components of the constructs and vectors of thepresent invention are well known to those skilled in the art. Suchtechniques include the use of linkers, such as synthetic linkers, forexample including one or more restriction enzyme sites.

One skilled in the art would appreciate that the term “operably linked”may encompass the association of nucleic acid sequences on a singlenucleic acid fragment so that the function of one is regulated by theother. For example, a promoter is operably linked with a coding sequencewhen it is capable of regulating the expression of that coding sequence(i.e., that the coding sequence is under the transcriptional control ofthe promoter). Coding sequences can be operably linked to regulatorysequences in a sense or antisense orientation.

Methods for transforming a plant according to the teachings of thepresent invention are known to those skilled in the art. As used hereinthe term “transformation” or “transforming” describes a process by whicha foreign DNA, such as a DNA construct, including expression vector,enters and changes a recipient cell into a transformed, geneticallyaltered or transgenic cell. Transformation may be stable, wherein thenucleic acid sequence is integrated into the organism genome and as suchrepresents a stable and inherited trait, or transient, wherein thenucleic acid sequence is expressed by the cell transformed but is notintegrated into the genome, and as such represents a transient trait.According to preferred embodiments the nucleic acid sequence of thepresent invention is stably transformed into the plant cell.

The genetically altered plants having altered content of the desiredsteroidal alkaloid(s) or steroidal saponin(s) according to the teachingsof the present invention are typically first selected based on theexpression of the gene or protein. Plants having enhanced or aberrantexpression of the gene or protein, are then analyzed for the content ofsteroidal alkaloids and optionally of steroidal saponins.

Detection of mutated GAME9, GAME11, BHLH, or GAME15 gene and/or thepresence of silencing molecule targeted to the gene and/orover-expression of the genes is performed employing standard methods ofmolecular genetics, known to a person of ordinary skill in the art.

For measuring the gene(s) or silencing molecule(s) expression, cDNA ormRNA should be obtained from an organ in which the nucleic acid isexpressed. The sample may be further processed before the detectingstep. For example, the polynucleotides in the cell or tissue sample maybe separated from other components of the sample, may be amplified, etc.All samples obtained from an organism, including those subjected to anysort of further processing are considered to be obtained from theorganism.

Detection of the gene(s) or the silencing molecule(s) typically requiresamplification of the polynucleotides taken from the candidate alteredorganism. Methods for DNA amplification are known to a person skilled inthe art. Most commonly used method for DNA amplification is PCR(polymerase chain reaction; see, for example, PCR Basics: frombackground to Bench, Springer Verlag, 2000; Eckert et al., 1991. PCRMethods and Applications 1:17). Additional suitable amplificationmethods include the ligase chain reaction (LCR), transcriptionamplification and self-sustained sequence replication, and nucleicacid-based sequence amplification (NASBA).

According to certain embodiments, the nucleic acid sequence comprisingthe GAME9, GAME11, BHLH, or GAME15 gene or its silencing moleculefurther comprises a nucleic acid sequence encoding a selectable marker.According to certain embodiments, the selectable marker confersresistance to antibiotic or to an herbicide; in these embodiments thetransgenic plants are selected according to their resistance to theantibiotic or herbicide.

Breeding

In some embodiments, transformation techniques including breedingthrough transgene editing, use of transgenes, use of transientexpression of a gene or genes, or use of molecular markers, or anycombination thereof, may be used in the breeding of a plant having analtered expression. If transformation techniques require use of tissueculture, transformed cells may be regenerated into plants in accordancewith techniques well known to those of skill in the art. The regeneratedplants may then be grown and crossed with the same or different plantvarieties using traditional breeding techniques to produce seed, whichare then selected under the appropriate conditions.

The content of steroidal alkaloids and/or steroidal saponins is measuredas exemplified hereinbelow and as is known to a person skilled in theart.

In some embodiments, an offspring plant comprises decreasedanti-nutritional contents or decreased toxins compared to at least oneof the progenitor plants. In some embodiments, an offspring plantcomprises improved resistance to a plant pathogen, pest, or predatorcompared to at least one of the progenitor plants.

In one embodiment, a plant as disclosed herein comprises a Solanaceaecrop plant. In some embodiments, a Solanaceae crop plant is selectedfrom the group consisting of Solanum lycopersicum, Solanum pennellii,Solanum tuberosum, Solanum chacoense, Capsicum annuum, and Solanummelongena. In some embodiments, a Solanaceae plant is selected from thegroup consisting of ground cherry, eggplant, potato, tomato, pepper,bell pepper, cayenne pepper, chili pepper, pimiento, tabasco pepper,tobacco, and bittersweet. In some embodiments, a Solanaceae plantcomprises any Solanaceae plant that produces a steroidal alkaloid or aglycosylated derivative thereof, or an unsaturated or saturatedsteroidal saponin or a glycoside derivative thereof, or any combinationthereof.

A skilled artisan would appreciate that plant breeding can beaccomplished through many different techniques ranging from simplyselecting plants with desirable characteristics for propagation, tomethods that make use of knowledge of genetics and chromosomes, to morecomplex molecular techniques.

A skilled artisan would appreciate that the term “hybrid plant” mayencompass a plant generated by crossing two plants of interest,propagating by seed or tissue and then growing the plants. When plantsare crossed sexually, the step of pollination may include crosspollination or self-pollination or back crossing with an untransformedplant or another transformed plant. Hybrid plants include firstgeneration and later generation plants. Disclosed herein is a method tomanipulate and improve a plant trait, for a non-limitingexample—increasing plant resistance, decreasing anti-nutritionalproperties in a plant, or decreasing toxins in a plant, or anycombination thereof.

Biomarkers

A skilled artisan would appreciate that the term “biomarker” comprisesany measurable substance in an organism whose presence is indicative ofa biological state or a condition of interest. In some embodiments, thepresence of a biomarker is indicative of the presence of a compound or agroup of compounds of interest. In some embodiments, the concentrationof a biomarker is indicative of the concentration of a compound or agroup of compounds of interest. In some embodiments, the concentrationof a biomarker is indicative of an organism phenotype.

Cellulose synthase like enzymes are hereby disclosed to have anessential role in the biosynthesis of steroidal alkaloids found inSolanaceae plants. Thus, in some embodiments, the expression level ofGAME15 is indicative of the capacity of a plant to produce steroidalalkaloids or glycosylated derivatives thereof, as well as α-tomatine anddehydrotomatine (e.g., in Solanum lycopersicum or tomato), α-chaconineand α-solanine (e.g., in Solanum tuberosum or potato), or α-solamargineand α-solasonine (e.g., in Solanum melongena or eggplant).

Further, one skilled in the art would appreciate that the term“comprising” used throughout is intended to mean that the geneticallymodified or gene edited plants disclosed herein, and methods of alteringexpression of genes, and altering production of SA and/or SGA withinthese genetically modified or gene edited plants includes the recitedelements, but not excluding others which may be optional. “Consistingof” shall thus mean excluding more than traces of other elements. Theskilled artisan would appreciate that while, in some embodiments theterm “comprising” is used, such a term may be replaced by the term“consisting of”, wherein such a replacement would narrow the scope ofinclusion of elements not specifically recited.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Materials and Methods Plant Material, Treatments and Generationof Transgenic Plants

Tomato (Solanum lycopersicum; cv. Micro Tom) and potato (Solanumliberosum; cultivar Desiree) plants were collected as describedpreviously (Itkin et al., 2001, supra). In potato, when the green partsstarted to dry, mature tubers (Stage 3) were collected, washed of soil,dried and kept at 4° C., at complete darkness.

The GAME9-silenced (RNAi) and overexpression (OX) constructs werecreated by introducing the corresponding GAME9 DNA fragments topK7GWIWG2(11) and pJCV52 binary vectors, respectively. Transgenic linesfor silencing and overexpression of GAME9 in tomato and potato weregenerated and tissue extracts were prepared and analyzed according toItkin et al. (2011, supra)

Table 1 below describes the oligonucleotides used for generation of theconstructs described herein. The GAME4-silencing (RNAi; GAME4i), GAME4overexpressing (GAME4oe) and GAME8-silencing constructs were generatedas described previously (Itkin et al., 2001, supra; WO 2012/095843).

TABLE 1 Oligonucleotides used for construct production SEQ NameSequence 5′ to 3′/Description ID NO. S107g043420AAAAAgaattcCGGATCTTCTCTCGAACTGGTCAA 20 EcoRI FwTo prepare GAME11 virus-induced gene silencing (VIGS) constructS107g043420 AAAAAgaattcCACTTTCATTGCTTCATCCATTAGATCT 21 EcoRI RvTo prepare GAME11 VIGS construct S107g043500AAAAAgaattcCTTAGCTTATGGCCACATCACACCTT 22 EcoRI FwTo prepare GAME18 VIGS construct S107g043500AAAAAgaattcACTCAAGATTTGGTGAAGCTGTGGTT 23 EcoRI RvTo prepare GAME18 VIGS construct GS-ForwardAAAAAGGCGCGCCAATCATAGAGAAGAAAGAAGACG 24 (Asa.)To constnict RNAi of GAME8 G8-Reverse (NotAAAAAGCGGCCGCACTCCTGCAGGAATTGTCATTTCTC 25 I) To construct RNAi of GAME8GAME9 RNAi aaaaaGCGGCCGCATGAGTATTGTAATTGATGATGATGAA 26 NotI Fw ATCTo construct RNAi of GAME9 GAME9 RNAi aaaaGGCGCGCCCACACGCCACAGATGGTTCTT27 AscI Rv To construct RNAi of GAME9 GAME9-TomGGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTATT 28 GW Fw GTAATTGATGATGATGAAATCTo pick up the gene from cDNA for overexpression (good for tomato)GAME9-Tom GGGGACCACTTTGTACAAGAAAGCTGGGTTCATACTAC 29 GW RvCTTCTGTCCTAAGCCTTo pick up the gene from cDNA for overexpression (good for tomato)GAME9-Pot GW GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATATT 30 FwGCAATTGATGATGATGATo pick up the gene from cDNA for overexpression (good for potato)GAME9-Pot GW GGGGACCACTTTGTACAAGAAAGCTGGGTTCATTTGTAT 31 RvCAACATTTGTAAATTCACACTo pick up the gene from cDNA for overexpression (good for potato)

Co-Expression Analysis

The tomato GAME1 (Solyc07g043490) and its potato ortholog SGT1(PGSC003DMG400011749) were used as ‘baits’ in the co-expressionanalysis, resulting in lists (sorted in descending order by r-value≥0.8)of co-expressed genes (for each ‘bait’ separately). Two homologous geneswere subsequently identified (Solyc12g006460 and PGSC0003DMG400024274 intomato and potato, respectively), which were highly correlated with the“bait” genes (r-value>0.9 in both species). Those genes were identifiedas GLYCOALKALOID METABOLISM 4 (GAME4, WO 2012/095843). The GAME4 geneswere further added as ‘baits’ to the previous (GAME1) co-expressionanalysis. The co-expression lists for GAME1 (SGT1) and GAME4 in bothspecies were used to construct co-expression correlation network. Theanalysis was performed as follows: tomato RNAseq transcriptome data fromdifferent tissues and organs (flesh, peel, seeds, roots, leaves, buds,flowers, pollen) and developmental stages (25 experiments in total)(Itkin et al., 2011, ibid) and potato RNAseq transcriptome data fromdifferent tissues and organs (40 experiments in total) (US2012/0159676), were used. First, an R script was used to performco-expression analysis (for each species) and the list of co-expressedgenes was constructed as a FASTA file, using a Perl script. Finally,BLASTa11 tools (Camacho C. et al., 2009. BMC Bioinform 10:421) were usedto find shared homologs between the two species. The tblastx criteriafor homolog similarity were set to p-value>0.05, minimum 25 nucleotides,and at least 60 percent similarity as an overall identity for each gene.The co-expression network was visualized with the Cytoscape program(Shannon P. et al., 2003. Genome Res. 13:2498-2504).

Phylogenetic Analysis

The protein sequences were aligned using the Muscle algorithm and thephylogenetic tree was analyzed and visualized by the SeaView v4.3.5program using the maximum likelihood method by PhyML 3.0(Expósito-Rodriguez M et al., 2008. BMC Plant Biol. 8:131) with thefollowing settings: model—LG; The approximate likelihood ratio test(aLRT) Shimodaira-Hasegawa-like (SH-like) procedure was used as astatistical test to calculate branch support (branch support—aLRT(SH-like)); invariable sites—optimized; across site ratevariation—optimized; tree searching operations—best for NNI & SPR;starting tree—BioNJ, optimize tree topology. The numbers on the branchesindicate the fraction of bootstrap iterations supporting each node. Theaccession numbers of the proteins used for the preparation of this treeand the organism names are listed in Table 2 hereinbelow; the tree ispresented in FIG. 12.

TABLE 2 Accession numbers of the sequences used for the construction ofthe phylogenetic tree Name as appears in FIG. 12 Latin and common nameAccession number GuCYP88D6 Glycyrrhiza uralensis BAG68929.1 LjCYP88D4Lotus japonicus BAG68927.1 MtCYP88D3 Medicago truncatula BAG68926.1CmCYP88A2 Cucurbita maxima AF212991 AtCYP88A3 Arabidopsis thalianaAAB71462.1 PsCYP88A7 Pisum sativum AAO23064.1 ZmCYP88A1 Zea maysNP_001105586.1 GmCYP88A26 Glycine max XP_003516638.1 CaCYP89A35 Capsicumannuum DQ114394 GmCYP89A36 Glycine max DQ340245 ZmCYP89B17 Zea maysCO465851.1 TmCYP89J1 Triticum monococcum AY914081 SlCYP88B1 (GAME4)Solanum lycopersicum Solyc12g006460.1.1 SpimpCYP88B1 (GAME4) Solanumpimpinellifolium contig 6356779 SpCYP88B1 (GAME4) Solanum pineliiAW618484.1, BG135958.1 StCYP88B2 (GAME4) Solanum tuberosum group PhurejaPGSC0003DMP400041994 StCYP88B1v2 (GAME4) Solanum tuberosum groupTuberosum PGSC0003DMP400041994 SlCYP88C2 Solanum lycopersicumSolyc10g007860.2.1 SmCYP88B3 (GAME4) Solanum melongena FS071104,FS071103 OsCYP90A3 Oryza sativa AC123526.1 SlCYP90A5 Solanumlycopersicum Solyc06g051750.2.1 ScCYP90A8 Citrus sinensis DQ001728.1ZeCYP90A11 Zinnia elegans BAE16977.1 PhCYP88C1 Petunia hybridaAAZ39647.1 AaCYP90A13 Artemisia annua ABC94481.1 AtCYP710A1 Arabidopsisthaliana AAC26690.1 SmCYP71A2 Solanum melongena X71654.1 GmCYP93E1Glycine max AB231332 HlCYP71C25 Hordeum lechleri AY462228 NtCYP71D16Nicotiana tabacum AF166332 MeCYP71E7 Manihot esculenta AY217351TaCYP71F1 Triticum aestivum AB036772 AoCYP71J1 Asparagus officinalisAB052131 MaCYP71N1v2 Musa acuminata AY062167 TaCYP72A6v1 Triticumaestivum AF123604 ZmCYP72A16 Zea mays AF465265 LeCYP72A51 Solanumlycopersicum Solyc10g051020.1.1 GmCYP72A61 Glycine max DQ340241MtCYP716A12 Medicago truncatula ABC59076.1 StCYP716A13 Solanum tuberosumPGSC0003DMP400013378 AaCYP716A14 Artemisia annua DQ363134 PsCYP716B2Picea sitchensis AY779543 SlCYP718A6 Solanum lycopersicumSolyc07g055970.1.1 MtCYP718A8 Medicago truncatula XP_003617455.1PsCYP719B1 Papaver somniferum EF451150 StCYP72A186 (GAME7) Solanumtuberosum PGSC0003DMG402012386 SlCYP72A186 (GAME7) Solanum lycopersicumSolyc07g062520 SlCYP72A188 (GAME6) Solanum lycopersicum Solyc07g043460StCYP72A188 (GAME6) Solanum tuberosum PGSC0003DMG400011750 GuCYP72A154Glycyrrhiza uralensis BAL45206.1 MtCYP72A59 Medicago truncatulaABC59078.1 NtCYP72A57 Nicotiana tabacum ABC69414.1 NtCYP72A54 Nicotianatabacum ABC69417.1 CrCYP72A1 Catharanthus roseus gi461812 MtCYP72A63Medicago truncatula gi371940452 NpCYP72A2 Nicotiana plumbaginifoliaAAB05376.3 SlCYP734A7 Solanum lycopersicum Solyc03g120060.1.1 StCYP72A29Solanum tuberosum BAB86912.1 StSYP72a56 Solanum tuberosumPGSC0003DMG400017325 StCYP72A208 (GAME8a) Solanum tuberosumPGSC0003DMG400026594 StCYP72A208 (GAME8b) Solanum tuberosumPGSC0003DMG400026586 SlCYP72A208 (GAME8a) Solanum lycopersicum TC243022SlCYP72A208 (GAME8b) Solanum lycopersicum SGN-U578058

Metabolite Analysis

Preparation of plant tissue extracts and profiling of semi-polarcompounds (including steroidal alkaloids and steroidal saponins) byUPLC-qTOF-MS and phytosterol content of the tomato leaves were carriedout as described previously (Itkin et al., 2011, supra).

Quantitative Real-Time PCR Assays

RNA was isolated and Quantitative Real-Time PCR was performed asdescribed previously (Itkin et al., 2011, supra). In addition, the TIP41gene (23) was used as an endogenous control for the potato samples.Oligonucleotides are listed in Table 1 hereinabove.

Production of Recombinant Enzyme

GAME 2, GAME17 and GAME18 were amplified from cDNA and subcloned intopACYCDUET-1 using BamH I and Pst I (GAME2, GAME18) or BamHI and XhoI(GAME17) restriction sites, and the insert was verified by sequencing.The resulting plasmids, pAC-GAME2/17/18 were transformed to E. coli BL21DE3. For expression of the GAME enzymes, fresh overnight cultures werediluted 1:100 in 25 ml 2×YT medium with 30 μg/ml chloramphenicol andincubated at 37° C. and 250 rpm until an A_(600 nm) of 0.4 was reached.Subsequently, IPTG was added to a concentration of 0.5 mM, and theincubation was continued overnight at 18° C. and 250 rpm. The next day,cells were harvested by centrifugation, and the pellet resuspended in 2ml of 50 mM Tris HCl pH=7.0, 15% glycerol, 0.1 mM EDTA and 5 mMβ-mercaptoethanol. After breaking the cells by sonication, insolublematerial was removed by centrifugation, and the soluble fractions wereused for characterization of the enzymes. Proteins were stored at −20°C. until further analysis.

Preparation of Substrates

For hydrolysis, 35 mg of α-tomatine was solved in 3 ml of 1 N HCl, andwas incubated for 15 min. at 100° C. Subsequently, the solution was puton ice, and NH₃ was added until the pH of the solution was 9.0. Thesolution was extracted with 4 ml water-saturated butanol. The butanolphase was evaporated to dryness under vacuum, the residual pellet solvedin 1 ml methanol and stored at −20° C. until further use. Thedegradation products of α-tomatine were separated on a Luna 5 μm C18(2)100 Å. LC Column 150×21.2 mm (Phenomenex, USA), using an isocraticelution with 25% acetonitrile in water and 0.1% formic acid. Compoundswere detected using a 3100 Mass Detector (Waters), and collected.Fractions were freeze-dried, and purity of compounds was verified byLC-MS. For identification of products, liquid chromatography, coupled toquadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was performedusing a Waters Alliance 2795 HPLC connected to a Waters 2996 PDAdetector and subsequently a QTOF Ultima V4.00.00 mass spectrometer(Waters, MS technologies, UK) operated in positive ionization mode. Thecolumn used was an analytical Luna 3 μm C18 (2) 100 Å; 150×2.0 mm(Phenomenex, USA) attached to a C18 pre-column (2.0×4 mm; AJO-4286;Phenomenex, USA). Degassed eluent A [ultra-pure water:formic acid(1000:1, v/v)] and eluent B [acetonitrile:formic acid (1000:1, v/v)]were used with flow rate of 0.19 ml/min. The gradient started at 5% Band increased linearly to 75% B in 45 min., after which the column waswashed and equilibrated for 15 min. before the next injection. Theinjection volume was 5 μl. This procedure yielded several milligrams ofpure γ-tomatine (tomatidine-galactoside-glucoside, T-Gal-Glu) andβ1-tomatine (tomatidine-galactoside-diglucoside. T-Gal-Glu-Glu).Tomatidine galactoside (T-Gal) could not be purified in this way due tostrong contamination with T-Gal-Glu. Therefore 5 mg tomatidine wasincubated with GAME1 and UDP-galactose in 1 ml reaction mix, asdescribed previously (Itkin et al., 2011, supra). T-Gal was purifiedfrom UDP-galactose by solid phase extraction. Waters OASIS HLB 3 cccolumns (Waters Corp., Milford, Mass.) was conditioned with 6 ml. 100%methanol followed by rinsing with 4 mL ultra-pure water. The reaction,supplemented with 10% methanol, was loaded and the cartridge wassubsequently washed with 4 mL ultra-pure water. Compounds were elutedwith 1 mL 75% methanol in ultra-pure water (v:v), and 0.4 mL 100%methanol. The solvent was removed from the combined eluate using a speedvacuum concentrator until a totally dry-pellet was obtained.

Enzyme Assays

The substrates T-Gal, and γ-tomatine were dissolved to 1 mM in 50% DMSO.Enzyme assays were carried out in 50 mM Tris HCl pH=7.0 containing 5 mMβ-mercaptoethanol using 5 μg/ml enzyme, 8 mM UDP-xylose and 0.02 mMsubstrate in a final reaction volume of 100 μl. After 2 h. of incubationunder agitation at 37° C., reactions were stopped by addition of 300 μlmethanol and 0.1% formic acid, and followed by brief vortexing andsonication for 15 min. Subsequently, the extracts were centrifuged for 5min. at 13,000 rpm and filtered through 0.45 μm filters (Minisart SRP4,Biotech GmbH, Germany), and analyzed by LC-MS (see above). The amount ofproduct was measured by the peak surface area in the LC-MS chromatogram,and compared to a control incubation in which an enzyme preparation ofan E. coli harboring an empty pACYCDUET-1. Masses used for detectionwere α-tomatine (C50H83NO21; m/z=1034.55 ([M+H]+)), β1-tomatine(C45H75NO17; m/z=902.51 ([M+H])), β2-tomatine (C44H73NO16; m/z=872.50([M+H]+)), γ-tomatine T-Gal-Glu (C39H65NO12; m/z=740.46 ([M+H])), andT-Gal (C33H55NO7; m/z=578.41 ([M+H])).

Virus Induced Gene Silencing (VIGS) Experiments

Vectors containing fragments of GAME genes were constructed and VIGSexperiments were conducted as described previously (Orzaea D et al.,2009. Plant Physiol. 150:1122-1134; Li R et al., 2006 J. Mass Spec.41:1-22). Plants infected with Agrobacterium, containing empty vectorand helper vector pTRV1, were used as control. Oligonucleotides used toprepare the pTRV2_DR_GW vectors are listed in Table 1 hereinabove.

Genome Sequence Analysis of the Wild Tomato Species

Partial genomic data obtained by re-sequencing (Dr. Arnaud G. Bovy,unpublished data) of three tomato wild species genomes (i.e. Solanumpennellii, S. pimpinelkfolium and S. chmielewskii) were analyzed for thepresence or absence of sequences (contigs) that align to the SGAsbiosynthesis gene clusters on tomato chromosomes 7 and 12. The TopHattoolkit (Trapnell C. 2012. Nat. Protoc. 7:562-578) was used for mappingreads of the wild species to the tomato genome (ITAG 2.4), as areference genome. The mapped reads were visualized with the IGV genomebrowser (Robinson J T et al., 2011. Nat. Biotechnol. 29:24-26). In orderto assemble and align the sequence of the contigs from the three wildspecies to the gene clusters on to the existing cultivated tomatosequences of chromosomes 7 and 12, a combination of the CLC workbench,CAPS BWA and SAMtools software packages and an in-house Perl script wereused.

Example 1 Genes Associated with SGA Biosynthesis

To discover genes associated with SGA biosynthesis, a co-expressionanalysis using transcriptome data from tomato and potato plants wasperformed. Coexpression with GAME1/SGT1 (chromosome 7) and GAME4(chromosome 12) as “baits” in either potato or tomato are presented in aform of a heatmap in Tables 3-6 herein below. Genes that are highlyco-expressed with either GAME1/SGT1 (chromosome 7) or GAME4 (chromosome12) are depicted with a large font and hold.

TABLE 3 Accession numbers, putative protein and co- expressionr-values - tomato, chromosome 7 r-value of correlation with tomato GAME1Gene name Putative protein expression Solyc07g043310 Aminotransferase−0.26 Solyc07g043320 Unknown Protein 0.12 Solyc07g043330 GRAS familytranscription factor 0.72 Solyc07g043340 Unknown Protein Solyc07g043350Unknown Protein Solyc07g043360 60S ribosomal protein L27 0.10Solyc07g043370 Transposase Solyc07g043380 Unknown Protein Solyc07g043390Cellulose synthase family protein 0.92 (GAME15) Solyc07g043400 UnknownProtein Solyc07g043410 UDP-xylose xylosyltransferase (GAME2)Solyc07g043420 2-oxoglutarate-dependent 0.79 dioxygenase Solyc07g043430Gag-Pol polyprotein Solyc07g043440 Glucosyltransferase-like proteinSolyc07g043450 Zeatin O-glucosyltransferase Solyc07g043460 CytochromeP450 (GAME 6) 0.91 Solyc07g043470 Unknown Protein Solyc07g043480UDP-glucose glucosyltransferase 0.88 Solyc07g043490UDP-glucosyltransferase family 1 1.00 protein (GAME1) Solyc07g043500UDP-glucosyltransferase 0.95 Solyc07g043510 Cysteine-type peptidase−0.24 Solyc07g043520 transposase Solyc07g043530 Unknown ProteinSolyc07g043540 Unknown Protein Solyc07g043550 UDP-arabinose 4-epimerase0.70 Solyc07g043560 Heat shock protein 4 0.24 Solyc07g043570 Aldo/ketoreductase family protein −0.09 Solyc07g043580 BHLH transcription factor0.43 Solyc07g043590 Amine oxidase family protein 0.03 Solyc07g043600Pentatricopeptide repeat-containing 0.43 protein Solyc07g043610 Auxinresponse factor 6 Solyc07g043620 Auxin response factor 6-1 0.65Solyc07g043630 Acyl-CoA synthetase/AMP-acid ligase II Solyc07g043640Acyl-CoA synthetase/AMP-acid ligase II Solyc07g043650 AMP-dependentsynthetase and ligase Solyc07g043660 Acyl-CoA synthetase/AMP-acid −0.16ligase II Solyc07g043670 Hydroxycinnamoyl CoA quinate transferase 2Solyc07g043680 Enoyl-CoA-hydratase Solyc07g043690 Enoyl-CoA-hydrataseSolyc07g043700 Acyltransferase

TABLE 4 Accession numbers, putative protein and co- expressionr-values - potato, chromosome 7 r-value of correlation with potato SGT1Gene name Putative protein expression PGSC0003DMG400011754 Gammaaminobutyrate −0.31 transaminase PGSC0003DMG400011753 Uro-adherencefactor A −0.40 PGSC0003DMG400011742 DELLA protein RGA 0.15PGSC0003DMG400011741 60S ribosomal protein L27 0.43 PGSC0003DMG400039612Conserved gene of unknown function PGSC0003DMG400011752 Cellulosesynthase 0.90 (GAME15) PGSC0003DMG400011740 beta-solanine rhamnosyl-0.90 transferase (SGT3) PGSC0003DMG400011751 2-oxoglutarate-dependent0.87 dioxygenase PGSC0003DMG400011750 Cytochrome P-450 0.92 (GAME 6)PGSC0003DMG400044993 Unknown Protein PGSC0003DMG400011749 solanidinegalactosyl- 1.00 transferase (SGT1) PGSC0003DMG402015928 OTU-likecysteine protease −0.24 family protein PGSC0003DMG401015928 Conservedprotein of −0.25 unknown function PGSC0003DMG400015927 UDP-arabinose4-epimerase −0.21 1 PGSC0003DMG400015920 Heat shock 70 kDa protein −0.17PGSC0003DMG402015926 Aldo/keto reductase −0.05 PGSC0003DMG401015926Isoform 2 of Transcription −0.33 factor PIF5 PGSC0003DMG400015925 Amineoxidase 0.11 PGSC0003DMG400015924 Pentatricopeptide repeat- 0.32containing protein PGSC0003DMG400015919 ARF8 0.07 PGSC0003DMG400036440AMP dependent ligase PGSC0003DMG400015923 Acyl:coA ligase acetate- coAsynthetase PGSC0003DMG400015922 Acyl:coA ligase acetate- coA synthetasePGSC0003DMG400044288 Acyltransferase PGSC0003DMG400015918Acyltransferase 0.03

TABLE 5 Accession numbers, putative protein and co- expressionr-values - tomato, chromosome 12 r-value of correlation with tomatoGAME4 Gene name Putative protein expression Solyc12g006530 Cycloartenolsynthase 0.08 Solyc12g006520 Cycloartenol synthase 0.05 Solyc12g006510Cycloartenol Synthase −0.12 Solyc12g006500 Phosphate translocatorprotein 0.15 Solyc12g006490 Beta-l-3-galaclosyl-o-glycosyl- 0.03glycoprotein Solyc12g006480 Nup205 protein 0.35 Solyc12g006470gamma-aminobutyrate Amino- 0.94 transferase-1ike protein Solyc12g006460Cytochrome P450 (GAME 4) 1.00 Solyc12g006450 gamma-aminobutyrate Amino-−0.13 transferase-like protein Solyc12g006440 Unknown Protein 0.25Solyc12g006430 UDP-glucuronosyltransferase 1- 1 82A1 Solyc12g006420Topoisomerase II-associated 0.08 protein PAT1 Solyc12g006410UDP-arabinse 4-epimerase Solyc12g006400 Unknown Protein Solyc12g0063902-oxoglutarate-dependent dioxygenase Solyc12g0063802-oxoglutarate-dependent 0.15 dioxygenase Solyc12g006370 Amine oxidasefamily protein −0.16 Solyc12g006360 Multidrug resistance protein mdtKSolyc12g006350 Auxin response factor 6 0.35 Solyc12g006340 Auxinresponse factor 6 0.47 Solyc12g006330 Acyltransferase-like proteinSolyc12g006320 ATP-dependent RNA helicase 0.14 Solyc12g006310Endoplasmic reticulum-Golgi 0.25 Solyc12g006300 WD-repeat protein-like−0.03 Solyc12g006290 Reticulon family protein 0.19 Solyc12g006280Myb-like DNA-binding protein

TABLE 6 Accession numbers, putative protein and co- expressionr-values - potato, chromosome 12 r-value of correlation with potatoGAME4 Gene name Putative protein expression PGSC0003DMG400020034Beta-amyrin synthase −0.13 PGSC0003DMG400024276 Beta-Amyrin Synthase−0.09 PGSC0003DMG400024277 Gene of unknown function 0.10PGSC0003DMG400024278 Phenylacetaldehyde 0.10 synthasePGSC0003DMG400024279 Conserved gene of −0.16 unknown functionPGSC0003DMG400024280 Triose phosphate/phosphate −0.06 translocator,non-green plastid, chloroplast PGSC0003DMG400024271 Acetylglucosaminyl-−0.06 transferase PGSC0003DMG400024273 Resistance protein PSH- 0.37 RGH6PGSC0003DMG400024281 Gamma aminobutyrate 0.94 transaminase isoform2PGSC0003DMG400024274 Cytochrome P450 1.00 monooxygenase GAME4PGSC0003DMG400024275 Gamma aminobutyrate 0.37 transaminase isoform3PGSC0003DMG400024282 Fortune-1 0.36 PGSC0003DMG400028806UDP-glycosyltransferase −0.18 82A1-like PGSC0003DMG401028807Topoisomerase II- associated protein PAT1 PGSC0003DMG402028807UDP-arabinse 4-epimerase PGSC0003DMG400028824 Gene of unknown functionPGSC0003DMG400028808 2-oxoglutarate-dependent −0.07 dioxygenasePGSC0003DMG400028809 2-oxoglutarate-dependent 0.61 dioxygenasePGSC0003DMG400028810 Amine oxidase −0.04 PGSC0003DMG400028825 MATEtransporter PGSC0003DMG400028826 Auxin response factor 6PGSC0003DMG400043090 Integrase core domain containing proteinPGSC0003DMG400037700 WRKY transcription factor 27 PGSC0003DMG400028811Acyltransferase PGSC0003DMG400028812 DEAD-box ATP-dependent 0.56 RNAhelicase 53 PGSC0003DMG400028814 WD-repeat protein −0.10PGSC0003DMG401028829 Polygalacturonase PGSC0003DMG400028815 Rebellionfamily protein 0.08 PGSC0003DMG400028830 Myb-like DNA-binding domain,SHAQKYF class family protein

Sixteen genes from each species were co-expressed with GAME1/SGT1 (Table7, FIG. 2). One of these genes, previously designated GLYCOALKALOIDMETABOLISM 4 (GAME4), encodes a member of the 88D subfamily ofcytochrome P450 proteins (FIG. 3). GAME4 and GAME1/SGT1 display a verysimilar expression profile in tomato and potato (WO 2010/095843). TheGAME1/SGT1 and GAME4 genes in tomato and potato are positioned inchromosomes 7 and 12 such that they are physically next to several oftheir co-expressed genes (FIG. 2).

A cluster of GAME1/SGT1 co-expressed genes spans a ˜200 Kbp genomicregion on chromosome seven. Together with GAME1, the tomato cluster iscomposed of 7 co-expressed genes. These include 3UDP-glycosyltransferases [GAME2 (termed SGT3 in potato); GAME17 andGAME18], a cytochrome P450 of the 72A subfamily (GAME6), a2-oxoglutarate-dependent dioxygenase (GAME11), and a cellulosesynthase-like protein (GAME15). It appears that in potato this clustercontains 5 co-expressed genes as it lacks homologs of the tomato genesencoding GAME17 and GAME18 UDP-glycosyltransferases. Enzyme activityassays were performed with the four recombinant clustered tomatoUDP-glycosyltransferases. GAME17 and GAME18 exhibitedUDP-glucosyltransferase activity when incubated with tomatidinegalactoside (T-Gal) and γ-tomatine (T-Gal-Glu) as a substrate,respectively, whereas GAME2 was shown to have an UDP-xylosyltransferaseactivity when incubated with β1-tomatine (T-Gal-Glu-Glu) as a substrate(FIGS. 4E, 4F, and 4G). GAMEI was previously shown to act as atomatidine UDP-galactosyltransferase in tomato (Itkin et al., 2011,supra). When incubating the 4 recombinant UGT enzymes in a single testtube, with tomatidine, and all glycoside donors (UDP-galactose, -glucoseand -xylose), the accumulation of the final SGA product α-tomatine wasobserved (FIG. 4H).

Two genes encoding putative transcription factors were identified amongthe genes co-expressed with GAME1/SGT1 and GAME4 (FIGS. 4A-4H): onegene, designated GAME9, was identified by the tomato ID Solyc01g090340and by the potato ID PGSC0003DMG400025989. It is described asethylene-responsive element binding factor 13 and contains a putativeAP2 domain. The other gene is the BHLH-transcription factor, identifiedby the tomato ID Solyc03g046570 and by the potato IDPGSC0003DMG400012262.

TABLE 7 Details of homologs co-expressed with known and putativesteroidal alkaloid- associated genes in both potato and tomato presentedin FIG. 2 Name Tomato ID Solyc Potato reads Tomato ID Extensin-likeprotein Solyc01g006400 PGSC0003DMG400023230 TCONS_00007692 GAME 9Solyc01g090340 PGSC0003DMG400025989 TCONS_00011729 Delta (24)-sterolreductase-like Solyc02g069490 PGSC0003DMG400021142 TCONS_00044548 BHLHtranscription factor Solyc03g046570 PGSC0003DMG400012262 TCONS_00055879LRR receptor-like protein kinase Solyc05g009100 PGSC0003DMG400014576TCONS_00101281 Glycosyltransferase Solyc05g053120 PGSC0003DMG402027210TCONS_00100675 Cellulose synthase-like (GAME15) Solyc07g043390PGSC0003DMG400011752 TCONS_00135034 GAME6 (CYP72) Solyc07g043460PGSC0003DMG400011750 TCONS_00137734 GAME1 (Galactosyltransferase)Solyc07g043490 PGSC0003DMG400011749 TCONS_00133014 GAME7 (CYP72)Solyc07g062520 PGSC0003DMG402012386 TCONS_00132326 (GAME1 r-value 0.66;(SGT1 r-value 0.63; GAME4 r-value 0.71) GAME4 r-value 0.73 ) Srt/Thrprotein kinase 6 Solyc08g066050 PGSC0003DMG400025461 TCONS_00151251Meiotic serine proteinase Solyc08g077860 PGSC0003DMG401012339TCONS_00149157 Sterol reductase Solyc09g009040 PGSC0003DMG400002720TCONS_00162820 Ubiquitin protein ligase Solyc10g008410PGSC0003DMG400021683 TCONS_00183263 Proteinase inhibitor IISolyc11g020960 PGSC0003DMG402003479 TCONS_00194999 GAME4 (CYP88)Solyc12g006460 PGSC0003DMG400024274 TCONS_00210154 Gamma-aminobutyrateAminotransferase-like Solyc12g006470 PGSC0003DMG400024281 protein(transaminase) (GAME12) Beta-solanine rhamnosyltransferase (SGT3) #N/APGSC0003DMG400011740 2-oxoglutarate-dependent dioxygenase (GAME11)Solyc07g043420 PGSC0003DMG400011751 GAME18 (Glycosyltransferase)Solyc07g043500 #N/A GAME17 (Glycosyltransferase) Solyc07g043480 #N/ATomato and potato sequences were obtained from Sol Genomics Network(solgenomics.net). r-value for co-expression ≥ 0.8. TCON number, acontig reference name given by the inventors in the assembly of RNAsecdata. N/A, not available.

Example 2 Functional Analysis of GAME9-Transcription Factor

GAME9-silencing (RNAi) and overexpressing (OX) constructs were createdby introducing the corresponding GAME9 DNA fragments to pK7GWIWG2(II)and pJCV52 binary vectors, respectively. Transgenic tomato and potatolines transformed with the respective GAME9 silencing and overexpressingconstructs were generated as previously described (Itkin et al., 2011,supra). Tissue extracts were prepared and analyzed as described in Itkinet al. (2011, supra).

The metabolic profiling of steroidal alkaloids using UPLC-TQ-MS wasperformed on extracts obtained from leaves and/or tubers of transgenicand wild type tomato and/or potato plants. In extract obtained frompotato tuber peels of potato lines in which the gene encoding GAME9 wassilenced (GAME9-RNAi lines) a reduction in α-solanine and α-chaconinewas observed (FIGS. 5A and 5B, respectively). Leaves from potatoGAME9-overexpression lines contained higher levels of α-solanine (FIG.5C) and α-chaconine (FIG. 5D) compared to the wild type. A similaraccumulation pattern was observed in potato leaves, having reducedamounts of α-chaconine and α-solanine in RNAi lines and increasedamounts of these steroidal alkaloids in lines overexpressing theGAME9-transcription factor (FIG. 6).

In tomato, leaves extract of a line overexpressing theGAME9-transcription factor (designated 5879) contained higher levels ofα-tomatine compared to its amount in leaf extract obtained from wildtype plants. On the contrary, down regulation of the expression ofGAME9-transcription factor (line 5871) resulted in significant reductionof α-tomatine content.

Example 3 Functional Characterization of the GAME Genes

GAME11 Silenced Plants

Virus induced gene silencing (VIGS) is a commonly used techniqueallowing systemic silencing of genes in various organs of the plant(Dinesh-Kumar S P et al., 2003. Methods Mol Diol 236:287-294).

Analysis of tomato leaves with VIGS-silenced GAME11, a putativedioxygenase in the cluster, revealed a significant reduction inα-tomatine levels and accumulation of several cholestanol-type steroidalsaponins.

Silencing of GAME11 dioxygenase in tomato results in depletion ofα-tomatine levels in leaves (m/z=1034.5) (FIG. 8A) while accumulatingcholestanol-type steroidal saponins [i.e. STSs; m/z=1331.6, 1333.6,1199.6, 1201.6 (major saponins)] (FIG. 88). FIG. 8C shows MS/MS spectrumof m/z=1331.6 (at 19.28 min.). FIG. 8D shows the fragmentation patternsof the saponin eluted at 19.28 min. and accumulating in GAME11-silencedleaves. The corresponding mass signals are marked with an asterisk onthe MS/MS chromatogram in FIG. 8C. The elemental composition andfragmentation patterns show that the compounds are cholestanol-typesaponins, lacking one hydroxy-group and the E-ring (in comparison tofurostanol-type saponins), which results in fragmentation, involvingmultiple losses of water molecules instead of tautomerisation andMcLafferty rearrangement of the E-ring.

GAME18 Silenced Plants

The role of GAME18 in creating the tetrasaccharide moiety of α-tomatinewas supported by Virus induced Gene Silencing (VIGS) assays asGAME18-silenced fruit accumulated γ-tomatine which was not present inthe control sample (FIGS. 9A-9E).

Among the metabolites extracted from GAME18-silenced mature green fruit,peaks of newly accumulating compounds were detected, corresponding tothe γ-tomatine standard (m/z=740.5) (FIGS. 9A-C), and γ-tomatinepentoside (m/z=872.5) (FIGS. 9D-9E).

GAME12 Silenced Plants

Silencing of GAME12 transaminase in tomato resulted in accumulation of afurastanol-type steroidal saponin (FIG. 4D). FIG. 10A shows thatGAME12-silenced leaves accumulate an STS (m/z=753.4), while it exists inonly minor quantities in wild type leaf FIG. 10B. FIG. 10C shows MS/MSspectrum of m/z=753.4 at 19.71 min. with interpretation of thefragments. Suggested structure of the STS at 19.71 min. is depicted inFIG. 10D, concluded from the characteristic mass fragments observed inthe MS/MS experiment.

Function of GAME7 and GAME8

Genes that were tightly co-expressed and positioned elsewhere in thegenome were also functionally examined. Two genes, designated GAME7 andGAME8 belong to the CYP72 subfamily of cytochrome P450s. GAME7 wasco-expressed in both species (potato and tomato) while StGAME8a andStGAME8b were strongly co-expressed with StSGT1 and StGAME4 in potato.At present, we could not demonstrate SGA-related activity for GAME7although as for GAME6 it was suggested to be involved in SGA metabolism(US 20120159676). Yet, GAME8-silenced tomato leaves accumulated22-(R)-hydroxycholesterol (FIGS. 11A-11D), a proposed intermediate inthe SGA biosynthetic pathway (FIG. 1). GAME8-silenced line accumulatesboth isomers in comparison to wild type (FIG. 11D). The (R)-isomer ismore abundant and hence most likely to be the substrate of GAME8.

FIG. 12 shows the phylogenetic tree of GAME genes in the plant CYP450protein family. The numbers on the branches indicate the fraction ofbootstrap iterations supporting each node.

Example 4 Proposed Biosynthetic Pathway in Solanaceous Plants

An expanded biosynthetic pathway in Solanaceous plants has beenproposed, as depicted in the schematic of FIG. 13 (dashed arrowsrepresent multiple enzymatic reactions in the pathway) with respect tothe tomato. This pathway can be broken down into four parts forconvenience. In Part I, a series of reactions (catalyzed, e.g., by SSR2,SMO3, SMO4) converts cylcloartenol to cholesterol. Byproducts includetriterpenoids and phytosterols. In Part II, a series of reactions(catalyzed, e.g., by GAME11, GAME6, GAME4, GAME12, GAME25) convertscholesterol to tomatidine (aglycone). Byproducts include steroidalsaponins (e.g., uttroside 13). In Part III, a series of reactions(catalyzed, e.g., by GAME1, GAME 17, GAME18, GAME2) converts tomatidineto steroidal glycoalkaloids (e.g., α-tomatine). In Part IV, a series ofreactions converts steroidal glycoalkaloids (e.g., α-tomatine) of agreen tomato to lycoperosides and/or esculeosides (e.g., esculeoside A)of a red tomato.

Example 5 Pathways Involving Steroidal Glycoalkaloid (SGA) Biosynthesisin Tomato, Potato, and Eggplant

A cellulose synthase-like gene (GAME15) in tomato, potato, and eggplanthas been identified as being associated with steroidal glycoalkaloid(SGA) biosynthesis (FIGS. 14A-14C). This gene has been shown to havebeen strongly co-expressed with other SGA biosynthesis genes (e.g.,GAME4, GAME12) and also with regulators of SGA biosynthesis (e.g.,GAME9).

Sequences were identified as follows:

>cellulose synthase like_tomato [SEQ ID NO: 32]ATGAAAAAAACCATGGAGCTCAACAAAAGCACTGTTCCACAACCTATCACCACCGTATACCGACTCCACATGTTCATCCACTCAATAATCATGCTTGCATTAATATACTACCGTGTATCTAATTTGTTTAAATTCGAAAACATTCTCAGTTTACAAGCACTTGCTTGGGCGCTCATCACTTTTGGTGAATTTAGTTTCATTCTCAAGTGGTTCTTCGGACAAGGTACTCGTTGGCGCCCCGTTGAACGAGATGTTTTCCCTGAAAACATTACTTGCAAAGATTCCGATCTACCGCCAATTGACGTAATGGTATTCACTGCCAATCCTAAGAAAGAGCCAATTGTAGATGTCATGAACACTGTGATATCCGCAATGGCTCTTGATTATCCCACCGATAAATTGGCTGTGTATCTCGCTGATGATGGAGGATGTCCATTGTCGTTGTACGCCATGGAACAAGCGTGTTTGTTTGCAAAGCTATGGTTACCTTTCTGTAGAAACTATGGAATTAAAACGAGATGCCCAAAAGCATTTTTTTCTCCGTTAGGAGATGATGACCGTGTTCTTAAGAATGATGATTTTGCTGCTGAAATGAAAGAAATTAAATTGAAATATGAAGAGTTCCAGCAGAAGGTGGAACATGCTGGTGAATCTGGAAAAATCAATGGTAACGTAGTGCCTGATAGAGCTTCGCTTATTAAGGTAATAAACGAGAGGGAGAACGAAAAGAGTGTGGATGATATGACGAAAATGCCCTTGCTAGTTTATGTATCCCGTGAAAGAAGATTCAACCGTCTTCATCATTTCAAGGGTGGATCTGCAAATGCTCTACTTCGAGTTTCTGGAATAATGAGTAATGCCCCCTATGTACTGGTGTTAGATTGTGATTTCTTCTGTCATGATCCAATATCAGCTAGGAAGGCAATGTGTTTTCATCTTGATCCAAAGCTATCATCTGATTTAGCCTATGTTCAGTTCCCTCAAGTCTTTTACAATGTCAGCAAGTCAGATATTTATGATGTCAAAATTAGACAGGCTTACAAGACAATATGGCATGGAATGGATGGTATCCAAGGCCCAGTGTTATCTGGGACTGGTTATTTTCTCAAGAGGAAAGCGTTATACACAAGTCCAGGAGTAAAAGAGGCGTATCTTAGTTCACCGGAAAAGCATTTTGGAAGGAGTAAAAGGTTTCTTGCTTCATTAGAGGAGAAAAATGGTTATGTTAAGGCAGATAAAGTCATATCAGAAGATATCATAGAGGAAGCTAAGATGTTAGCTACTTGTGCATATGAGGATGGCACACATTGGGGTCAAGAGATTGGTTATTCATACGATTGTCATTTGGAGAGCACTTTTACTGGTTATCTATTACACTGCAAAGGGTGGACATCTACTTATTTGTATCCAGACAGGCCATCTTTCTTGGGTTGTGCCCCAGTTGATATGCAAGGTTTCTCATCACAGCTCATCAAATGGGTTGCTGCACTTACACAAGCTGGTTTATCACATCTCAATCCCATCACTTATGGTTTGAGTAGTAGGATGAGGACTCTCCAATGCATGTGCTATGCCTATTTGATGTATTTCACTCTTTATTCTTGGGGAATGGTTATGTATGCTAGTGTTCCTTCTATTGGCCTTTTGTTTGACTTCCAAGTCTATCCTGAGGTACATGATCCGTGGTTTGCAGTGTATGTGATTGCTTTCATATCGACAATTTTGGAGAATATGTCGGAGTCAATTCCAGAAGGGGGATCAGTTAAAACGTGGTGGATGGAATACAGGGCATTGATGATGATGGGAGTTAGCGCAATATGGTTAGGAGGATTGAAAGCTATATATGACAAGATAGTCGGAACACAAGGAGAGAAATTGTATTTGTCGGACAAGGCAATTGACAAGGAAAAGCTCAAGAAATACGAGAAGGGCAAATTTGATTTCCAAGGAATAGGGATACTTGCTCTGCCACTGATAGCATTTTCCGTGTTGAACCTCGTAGGCTTCATTGTTGGAGCTAATCATGTCTTTATTACTATGAACTACGCAGGCGTGCTGGGCCAACTCCTCGTATCATCGTTCTTCGTCTTTGTTGTCGTCACTGTTGTCATTGATGTTGTATCTTTCTTAAAGGTTTCTTAA >cellulose synthase like_tomato[SEQ ID NO: 33]MKKTMELNKSTVPQPITTVYRLHMFIHSIIMLALIYYRVSNLFKFENILSLQALAWALITFGEFSFILKWFFGQGTRWRPVERDVFPENITCKDSDLPPIDVMVFTANPKKEPIVDVMNTVISAMALDYPTDKLAVYLADDGGCPLSLYAMEQACLFAKLWLPFCRNYGIKTRCPKAFFSPLGDDDRVLKNDDFAAEMKEIKLKYEEFQQKVEHAGESGKINGNVVPDRASLIKVINERENEKSVDDMTKMPLLVYVSRERRFNRLHHFKGGSANALLRVSGIMSNAPYVLVLDCDFFCHDPISARKAMCFHLDPKLSSDLAYVQFPQVFYNVSKSDIYDVKIRQAYKTIWHGMDGIQGPVLSGTGYFLKRKALYTSPGVKEAYLSSPEKHFGRSKRFLASLEEKNGYVKADKVISEDIIEEAKMLATCAYEDGTHWGQEIGYSYDCHLESTFTGYLLHCKGWTSTYLYPDRPSFLGCAPVDMQGFSSQLIKWVAALTQAGLSHLNPITYGLSSRMRTLQCMCYAYLMYFTLYSWGMVMYASVPSIGLLFDFQVYPEVHDPWFAVYVIAFISTILENMSESIPEGGSVKTWWMEYRALMMMGVSAIWLGGLKAIYDKIVGTQGEKLYLSDKAIDKEKLKKYEKGKFDFQGIGILALPLIAFSVENLVGFIVGANHVFITMNYAGVLGQLLVSSFFVFVVVTVVID VVSFLKVS>cellulose synthase like_solanum pennellii [SEQ ID NO: 34]ATGAAAAAAACCATGGAGCTCAACAAAAGCACTGTTCCACAACCTATCACCACCGTATACCGACTCCACATGTTCATCCACTCAATAATCATGCTTGCATTAATATACTACCGTGTATCTAATTTGTTTAAATTCGAAAACATTCTCAGTTTACAAGCACTTGCTTGGCTACTCATCACTTTTGGTGAATTTAGTTTCATTCTCAAGTGGTTCTTCGGACAAGGAACTCGTTGGCGCCCCGTTGAACGAGATGTTTTCCCTGAAAACATTACTTGCAAAGATTCCGATCTACCGCCAATTGACGTAATGGTGTTCACTGCCAATCCTAAGAAAGAGCCAATTGTAGATGTCATGAACACTGTGATATCCGCAATGGCTCTTGATTATCCCACCGATAAATTGGCTGTGTATCTGGCCGATGATGGAGGATGTCCATTGTCCTTGTACGCCATGGAACAAGCATGTTTGTTTGCAAAGCTATGGTTACCTTTCTGTAGAAAGTATGGAATTAAAACGAGATGCCCAAAAGCATTTTTTTCTCCGTTAGGAGATGATGACCGTGTTCTTAAGAATGATGATTTTGCTGCTGAAATGAAAGAAATTAAATTGAAATATGAAGAGTTCCAGCAGAACGTGGAACATGCTGGTGAATCTGGAAAAATCAATGGCAACGTAGTGCCTGACAGAGCTTCGCTTATTAAGGTAATAAACGAGAGGGAGAACGAAAAGAGTGTCGATGATTTAACGAAAATGCCCTTGCTAGTTTATGTATCCCGTGAAAGAAGATTCAACCGTCTTCATCATTTCAAGGGTGGATCTGCAAATGCTCTACTTCGAGTTTCTGGAATAATGAGTAATGCCCCCTATGTACTGGTGTTAGATTGTGATTTCTTCTGTCATGATCCGATATCAGCTAGGAAAGCAATGTGTTTTCATCTTGATCCAAAGCTATCATCTGATTTAGCCTATGTTCAGTTCCCTCAAGTCTTTTACAATGTCAGCAAGTCCGATATTTATGATGTCAAAATTAGACAGGCTTACAAGAGAATATGGCATGGAATGGATGGTATCCAAGGCCCAGTGTTATCTGGAACTGGTTATTTTCTCAAGAGGAAGGCGTTATACACAAGTCCAGGAGTAAAAGAGGCGTATCTTAGTTCACCGGAAAAGCATTTTGGAAGGAGTAAAAAGTTCCTTGCTTCATTAGAGGAGAAAAATGGTTATGTTAAGGCAGATAAAGTCATATCAGAAGATATCATAGAGGAAGCTAAGATCTTAGCTACTTGTGCATATGAGGATGGCACACATTGGGGTCAAGAGATTGGTTATTCATACGATTGTCATTTGGAGAGCACTTTTACTGGTTATCTATTACACTGCAAAGGGTGGACATCTACTTATTTGTATCCAGACAGGCCATCTTTCTTGGGTTGTGCCCCAGTTGATATGCAAGGTTTCTCATCACAGCTCATAAAATGGGTTGCTGCACTTACACAAGCTGGTCTATCACATCTCAATCCCATCACTTATGGTTTGAGTAGTAGGATGAGAACTCTCCAATGCATGTGCTATGCCTATTTGATGTATTTCACTCTTTATTCTTGGGGAATGGTTATGTATGCTAGTGTTCCTTCTATTGGCCTTTTGTTTGGCTTCCAAGTCTACCCTGAGGTACATGATCCATGGTTTGCAGTGTATGTGATTGCTTTCATATCGACAATTTTGGAGAATATGTCGGAGTCAATTCCAGAAGGGGGATCAGITAAAACGTGGTGGATGGAATACAGGGCATTGATGATGATGGGAGTTAGCGCAATATGGTTAGGAGGATTGAAAGCTATATATGACAAGATAGTCGGAACACAAGGAGAGAAATTGTATTTGTCGGACAAGGCAATTGACAAGGAAAAGCTCAAGAAATACGAGAAGGGCAAATTTGATTTCCAAGGAATAGGGATACTTGCTCTGCCATTGATAGCATTTTCCGTGTTGAACCTCGTAGGCTTCATTGTTGGAGCTAATCATGTCTTTATTACTATGAACTACGCAGGCGTGCTGGGCCAACTCCTCGTATCATCATTCTTCGTCTTTGTTGTCGTCACTGTTGTCATTGATGTTGTATCTTTCTTAAAGGTTTCTTAA>cellulose synthase like_solanum pennellii [SEQ ID NO: 35]MKKTMELNKSTVPQPITVYRLHMFLHSIIMLALIYYRVSNLFKFENILSLQALAWLLITFGEFSFILKWFFGQGTRVVRPVERDVFPENITCKDSDLPPIDVMVFTANPKKEPIVDVMNTVISAMALDYPTDKLAVYLADDGGCPLSLYAMEQACLFAKLWLPFCRKYGIKTRCPKAFFSPLGDDDRVLKNDDFAAEMKEIKLKYEEFQQNVEHAGESGKINGNVVPDRASLIKVINERENEKSVDDLTKMPLLVYVSRERRFNRLHHFKGGSANALLRVSGIMSNAPYVLVLDCDFFCHDPISARKAMCFHLDPKLSSDLAYVQFPQVFYNVSKSDIYDVKIRQAYKTIWHGMDGIQGPVLSGTGYFLKRKALYTSPGVKEAYLSSPEKHFGRSKKFLASLEEKNGYVKADKVISEDIIEEAKILATCAYEDGTHWGQEIGYSYDCHLESTFTGYLLHCKGWTSTYLYPDRPSFLGCAPVDMQGFSSQLIKWVAALTQAGLSHLNPITYGLSSRVIRTLQCMCYAYLMYFTLYSWGMVMYASVPSIGLLFGFQVYPEVHDPWFAVYVIAFISTILENMSESIPEGGSVKTWWMEYRALMMMGVSAIWLGGLKAIYDKIVGTQGEKLYLSDKAIDKEKLKKYEKGKFDFQGIGILALPLIAFSVLNLVGFIVGANHVFITMNYAGVLGQLLVSSFFVFVVVTVVIDVVSFLKVS >cellulose synthase like_potato [SEQ ID NO: 36]ATGAAAAAAACCATGGAGCTCAACAAAAGCACTGTTCCACAACCTATCACCACCATATACCGACTCCACATGTTTATCCACTCTATAATCATGGTTGCATTAATATACTACCGTGTATCTAATTTGTTTAAATTCGAAAACATTCTGAGTTTACAAGCACTTGCTTGGGTACTCATCACTTTTGGTGAATTTAGTTTCATTCTCAAGTGGTTCTTCGGACAAGGAACTCGTTATCGCCCTGTTGAAAGAGATGTTTTCCCTGAAAACATAACTTGCAAAGATTCCGATCTACCACCAATTGACGTAATGGTATTCACTGCCAATCCTAAGAAAGAGCCAATTGTGGATGTCATGAACACTGTGATATCCGCAATGGCTCTTGATTATCCTACGGATAAATTGGCTGTGTATCTGGCTGATGATGGAGGATGTCCTTTGTCATTGTACGCCATGGAAGAAGCATGTGTGTTTGCAAAGCTGTGGCTACCTTTCTGTAGGAAGTATGGAATTAAAACTAGATGCCCTAAAGCGTTTTTTTCTCCTTTAGGAGATGATGAACGTGTTCTTAAGAATGATGATTTTGATGCTGAAATGAAAGAAATTAAATTGAAATATGAAGAGTTCCAGCAGAATGTGGAACGTGCTGGTGAATCTGGAAAAATCAATGGTAACGTAGTGCCTGATAGAGCCTCGTTTATTAAGGTAATAAACGACAGAAAAGCGGAGAGCGAAAAGAGTGCCGATGATTTAACGAAAATGCCCTTGCTAGTTTATGTATCCCGTGAAAGAAGATTCAACCGTCTTCATCACTTCAAGGGTGGATCTGCAAATGCTCTTCTTCGAGTTTCTGGAATAATGAGTAATGCCCCCTATATACTGGTGTTAGATTGTGATTTCTTCTGTCATGATCCAATATCAGCTAGGAAGGCAATGTGTTTTCATCTTGATCCAAAGCTATCATCTGATTTAGCTTATGTTCAGTTCCCTCAAGTCTTTTACAATGTCAGCAAGTCCGATATTTATGATGTCAAAATTAGACAGGCTTACAAGACAATATGGCATGGAATGGATGGTATCCAAGGCCCAGTGTTATCAGGAACTGGTTATTTTCTGAAGAGGAAGGCGTTATACACGAGTCCAGGAGTAAAGGAGGAGTATCTTAGTTCACCGGAAAAGCATTTTGGAAGGAGTAAAAAGTTCCTTGCTTCACTAGAGGAGAAAAATGGTTATGTTAAGGCAGAGAAAGTCATATCAGAAGATATCGTAGAGGAAGCTAAGACCTTAGCTACTTGTGCATATGAGGATGGCACACATTGGGGTCAAGAGATTGGTTATTCATACGATTGTCATTTGGAGAGCACTTTTACTGGTTATCTATTACACTGCAAAGGGTGGAGATCGACTTATTTGTATCCAGACAGGCCATCTTTCTTGGGTTGTGCCCCAGTTGATATGCAAGGTTTCTCCTCACAGCTCATAAAATGGGTTGCTGCACTTACACAAGCTGGTTTATCACATCTCAATCCCATCACTTATGGCTTTAGTAGCAGGATGAAAACTCTCCAATGCATGTGCTATGCCTATTTGATATATTTCACTCTTTATTCTTGGGGAATGGTTCTATATGCTAGTGTTCCTTCTATTGGCCTTTTGTTTGGCTTCCAAGTCTATCCCGATGTACATGATCCATGGTTTGCAGTGTATGTGATTGCTTTCATATCGGCAATTTTGGAGAATATGTCGGAGTCAATTCCTGATGGGGGATCATTTAAATCTTGGTGGATGGAATACAGGGCACTGATGATGATGGGAGTTAGTGCAATATGGTTAGGAGGATTGAAAGCTATATTAGACAGGATAATCGGAACAGAAGGAGAGAAATTGTATTTATCGGACAAGGCAATTGACAAGGAAAAGCTCAAGAAATACGAGAAGGGGAAATTTGATTTCCAAGGAATAGGGATACTTGCTGTACCATTGATAGCATTTTCCTTGTTGAACCTCGTAGGCTTCATTGTTGGAGCTAATCATGTCTTTATTACTATGAACTACGCAGGTGTGCTTGGCCAACTCCTCGTATCATCCTTCTTCGTCTTTGTCGTGGTCACTGTTGTCATTGATGTCGTTTCTTTCTTAAAGGTTTCTTAA >cellulose synthase like_potato[SEQ ID NO: 37]MELNKSTVPQPITTIYRLHMFIHSIIMVALIYYRVSNLFKFENILSLQALAWVLITFGEFSFILKWFFGQGTRYRPVERDVFPENITCKDSDLPPIDVMVFTANPKKEPIVDVMNTVISAMALDYPTDKLAVYLADDGGCPLSLYAMEEACVFAKLWLPFCRKYGIKTRCPKAFFSPLGDDERVLKNDDFDAEMKEIKLKYEEFQQNVERAGESGKINGNVVPDRASFIKVINDRKAESEKSADDLTKMPLLVYVSRERRFNRLHHFKGGSANALLRVSGIMSNAPYILVLDCDFFCHDPISARKAMCFHLDPKLSSDLAYVQFPQVFYNVSKSDIYDVKIRQAYKTIWHGMDGIQGPVLSGTGYFLKRKALYTSPGVKEEYLSSPEKHFGRSKKFLASLEEKNGYVKAEKVISEDIVEEAKTLATCAYEDGTHWGQEIGYSYDCHLESTFTGYLLHCKGWRSTYLYPDRPSFLGCAPVDMQGFSSQLIKWVAALTQAGLSHLNPITYGFSSRMKTLQCMCYAYLIYFTLYSWGMVLYASVPSIGLLFGFQVYPDVHDPWFAVYVIAFISAILENMSESIPDGGSFKSWWMEYRALMMMGVSAIWLGGLKAILDRIIGTEGEKLYLSDKAIDKEKLKKYEKGKFDFQGIGILAVPLIAFSLLNLVGFIVGANHVFITMNYAGVLGQLLVSSFFVFVVVTVVIDVVSFLKVS>cellulose synthase like_solanum chacoense [SEQ ID NO: 38]ATGAAAAAAACCATGGAGCTCAACAAAAGCACTGTTCCACAACCTATCACCACCATATACCGACTCCACATGTTCGTCCATTCTATAATCATGGCTGCATTAATATACTACCGTGTATCTAATTTGTTTAAATTCGAAAACATTCTGAGTTTACAAGCACTTGCTTGGGTACTCATCACTTTTGGTGAATTTAGTTTCATTCTCAAGTGGTTCTTCGGACAAGGAACTCGTTGGCGCCCTGTTGAAAGAGATGTTTTCCCTGAAAACATAACTTGCAAAGATTCCGATCTACCACCAATTGACGTAATGGTATTCACTGCCAATCCTAAGAAAGAGCCAATTGTGGATGTCATGAACACTGTGATATCCGCAATGGCTCTAGATTATCCTACGGATAAATTGGCTGTGTATCTGGCTGATGATGGAGGATGTCCTTTGTCATTGTACGCCATGGAAGAAGCATGTGTGTTTGCAAAGCTGTGGCTACCTTTCTGTAGGAAGTATGGAATTAAAACCAGATGCCCTAAAGCGTTTTTTTCTCCTTTAGGAGATGATGACCGTGTTCTTAAGAATGATGATTTTGATGCTGAAATGAAAGAAATTAAATTGAAATATGAAGAGTTCCAGCAGAATGTGGAACGTGCTGGTGAATCTGGAAAAATCAATGGTAACGTAGTGCCTGATAGAGCCTCGTTTATTAAGGTAATAAACGACAGAAAAACGGAGAGCGAAAAGAGTGCCGATGATTTAACGAAAATGCCCTTGCTAGTTTATGTATCCCGTGAAAGAAGATTCAACCGTCTTCATCACTTCAAGGGTGGATCTGCAAATGCTCTTCTTCGAGTTTCTGGAATAATGAGTAATGCCCCCTATATACTGGTGTTAGATTGTGATTTCTTCTGTCATGATCCAATATCAGCTAGGAAGGCAATGTGTTTTCATCTTGATCCAAAGCTATCATCTGATTTAGCTTATGTTCAGTTCCCTCAAGTCTTTTACAATGTCAGCAAGTCCGATATTTATGATGTCAAAATTAGACAGGCTTACAAGACAATATGGCATGGAATGGATGGTATCCAAGGCCCAGTGTTATCAGGAACTGGTTATTTTCTGAAGAGGAAGGCGTTATACACGAGTCCAGGAGTAAAGGAGGAGTATCTTAGTTCACCGGAAAAGCATTTTGGAAGGAGTAAAAAGTTCCTTGCTTCACTAGAGGAGAAAAATGGTTATGTTAAGGCAGAGAAAGTCATATCAGAAGATATCGTAGAGGAAGCTAAGACCTTAGCTACTTGTGCATATGAGGATGGTACACATTGGGGTCAAGAGATCGGTTATTCATACGATTGTCATTTGGAGAGCACTTTTACTGGTTATCTATTACACTGCAAAGGGTGGACATCGACTTATTTGTATCCAGACAGGCCATCTTTCTTGGGTTGTGCTCCAGTTGATATGCAAGGTTTCTCCTCACAGCTCATAAAATGGGTTGCTGCACTTACACAAGCTGGTTTATCACATCTCAATCCCATCACTTATGGCTTGAGTAGCAGGATGAAAACTCTCCAATGCATGTGCTATGCCTATTTGATATATTTCACTCTTTATTCTTGGGGAATGGTTCTATATGCTAGTATTCCTTCTATTGGTCTTTTGTTTGGCTTCCAAGTCTATCCGGAGGTACATGATCCATGGTTTGCAGTGTATGTGATTGCTTTCATATCGACAATTTTGGAGAATATGTCGGAGTCAATTCCAGAAGGGGGATCATTTAAATCGTGGTGGATGGAATACAGGGCACTGATGATGATGGGAGTTAGTGCAATATGGTTAGGAGGATTGAAAGCTATATTAGACAAGATAATCGGAACAGAAGGAGAGAAATTGTATTTGTCAGACAAGGCAATTGACAAGGAAAAGCTCAAGAAATACGAGAAGGGGAAATTTGATTTCCAAGGAATAGGGATACTTGCTGTACCATTGATAGCATTTTCCCTGTTGAACCTGGTAGGCTTCATTGTTGGAGCTAATCATGTCTTTATTACTATGAACTACGCAGGTGTGCTTGGCCAACTCCTCGTATCATCCTTCTTCGTCTTTGTCGTGGTCACTGTTGTCATTGATGTCGTTTCTTTCTTAAAGGTTTCTTAA>cellulose synthase like_solanum chacoense [SEQ ID NO: 39]MKKTMELNKSTVPQPITTIYRLHVIFVHSIIMAALIYYRVSNLFKFENILSLQALAWVLITFGEFSFILKWFFGQGTRWRPVERDVFPENITCKDSDLPPIDVMVFTANPKKEPIVDVMNTVISAMALDYPTDKLAVYLADDGGCPLSLYAMEEACVFAKLWLPFCRKYGIKTRCPKAFFSPLGDDDRVLKNDDFDAEMKEIKLKYEEFQQNVERAGESGKINGNVVPDRASFIKVINDRKTESEKSADDLTKMPLLVYVSRERRFNRLHHFKGGSANALLRVSGIMSNAPYILVLDCDFFCHDPISARKAMCFHLDPKLSSDLAYVQFPQVFYNVSKSDIYDVKIRQAYKTIWHGMDGIQGPVLSGTGYFLKRKALYTSPGVKEEYLSSPEKHFGRSKKFLASLEEKNGYVKAEKVISEDIVEEAKTLATCAYEDGTHWGQEIGYSYDCHLESTFTGYLLHCKGWTSTYLYPDRPSFLGCAPVDMQGFSSQLIKWVAALTQAGLSHLNPITYGLSSRMKTLQCMCYAYLIYFTLYSWGMVLYASIPSIGLLFGFQVYPEVHDPWFAVYVIAFISTILENMSESIPEGGSFKSWWMEYRALMMMGVSAIWLGGLKAILDKIIGTEGEKLYLSDKAIDKEKLKKYEKGKFDFQGIGILAVPLIAFSLLNLVGFIVGANHVFITMNYAGVLGQLLVSSFFVFVVVTVVIDVVSFLKVS >cellulose synthase like_eggplant [SEQ ID NO: 40]ATGAAAAAACAAATGGAGCTCAACAGAAGTGTTGTACCGCAACCTATCACCACCATTTACCGTCTCCACATGTTTATCCATGCCCTAATCATGCTAGCACTAATATACTACCGTGTCTCTAATTTGGCCAAATTCGAAAACATCCTCAGTTTACAAGCACTTGCTTGGGCTCTTATCACGTTAGGTGAACTTTGTTTCATAGTCAAGTGGTTCTTCGGACAAGGGACTCGTTGGCGTCCTGTTGATAGGGATGTCTTCCCTGAAAACATCACTTGTCCAGATTCCGAGCTACCCCCCATTGATGTCATGGTTTTCACTGCAAATCCTAAGAAAGAGCCAATTGTGGATGTCATGAACACTGTCATATCCGCAATGGCTCTTGATTACCCGACCGACAAATTGGCCGTTTATTTGTCTGATGATGGAGGATGCCCCTTGACGTTGTACGCAATGGAGGAAGCTTGTTCCTTTGCCAAGTTGTGGCTACCTTTTTGTAGGAAGTATGGAATCAAAACAAGGTGCCCTAAGGCGTTTTTTTCTCCATTAGGAGAAGATGACCGTGTATTGAAGAGTGATGACTTTGTTTCTGAAATGAAAGAAATGAAGTCAAAATATGAAGAGTTCCAGCAGAACGTGGACCGTGCTGGTGAATCCGGAAAAATCAAAGGTGACGTAGTGCCTGATAGACCCGCGTTTCTTAAGGTACTAAATGACAGGAAGACGGAGAACGAGAAGAGTGCAGACGATTTAACTAAAATGCCTTTGCTAGTATACGTATCCCGTGAAAGAAGAACTCACCGTCGCCATCACTTCAAGGGTGGATCTGCAAATGCTCTTCTTCGAGTTTCTGGGATAATCAGTAATGCCCCCTATATACTGGTTTTAGATTGTGATTTCTTCTGTCATGATCCAATATCAGCTCGGAAGGCAATGTGTTTCCATCTTGATCCAAAACTATCACCTGACTTAGCTTACGTGCAGTTCCCTCAAGTGTTTTACAATGTTAGCAAGTCCGATATTTACGACGTCAAAATTAGACAGGCTTACAAGACAATATGGCACGGGATGGATGGTATCCAAGGCCCAGTGTTATCGGGAACTGGTTATTTTTTAAAAAAGAAGGCGTTGTACACGAGTCCAGGTCTAAAAGATGAGTATCTTAGTTCACCGGAAAAGCATTTCGGAACGAGTAGAAAGTTCATTGCTTCACTAGAGGAGAATAATTATGTTAAGCAAGAGAAAGTCATATCAGAAGATATCATAGAGGAAGCTAAGAGACTGGCTACTTGTGCATACGAGGATGGCACACATTGGGGTCAAGAGGCAAACAGGCCATCTTTCTTGGGTTGTGCCCCAGTTGATATGCAAGGTTTCTCCTCACAGCTCATAAAATGGGTTGCTGCACTCACACAAGCAGGTCTATCACATCTCAATCCCATCACTTACGGCTTCAAGAGCAGAATGAGAACTCTCCAAGTCTTGTGTTATGCCTATTTGATGTATTTCTCTCTTTATTCTTGGGGAATGGTTCTACATGCTAGTGTTCCTTCTATTGGCCTTCTCTCTGGCATTAAAATCTACCCGGAGGTGTATGATCCATGGTTTGTTGTGTATGTGATTGCTTTCATATCAACAATTTTGGAGAATATGTCGGAATCAATTCCGGAAGGGGGATCGGTTAAAACGTGGTGGATGGAATACAGGGCACTGATGATGATGGGAGTTAGTGCAATATGGCTAGGAGGAGTGAAAGCCATAGTAGACAAGATCATCGGAACGCAAGGAGAGAAATTGTATTTGTCGGACAAAGCAATTGACAAGGAAAAGCTCAAGAAATACGAGAAGGGGAAATTTGATTTCCAAGGAATAGGAATACTTGCTGTACCATTGATAACATTTTCTGTGTTGAACCTGGTAGGCTTCTTGGTTGGAATTAATCAAGTGTTGATAACGATGAAGTTCGCAGGCGTGCTGGGCCAACTCCTCGTATCATCCTTCTTCGTCTTTGTCGTCGTTACTGTTGTCATTGATGTCGTATCTTTCTTAAAGGATTCTTAA >cellulose synthase like_eggplant [SEQ ID NO: 41]MKKQMELNRSVVPQPITTIYRLHMFIHALIMLALIYYRVSNLAKFENILSLQALAWALITLGELCFIVKWFFGQGTRWRPVDRDVFPENITCPDSELPPIDVMVFTANPKKEPIVDVMNTVISAVIALDYPTDKLAVYLSDDGGCPLTLYAMEEACSFAKLYVLPFCRKYGIKTRCPKAFFSPLGEDDRVLKSDDFVSEMKEMKSKYEEFQQNVDRAGESGKIKGDVVPDRPAFLKVLNDRKTENEKSADDLTKMPLLVYVSRERRTHRRHHFKGGSANALLRVSGIISNAPYILVLDCDFFCHDPISARKAMCFHLDPKLSPDLAYVQFPQVFYNVSKSDIYDVKIRQAYKTIWHGMDGIQGPVLSGTGYFLKKKALYTSPGLKDEYLSSPEKHFGTSRKFIASLEENNYVKQEKVISEDIIEEAKRLATCAYEDGTHWGQEANRPSFLGCAPVDMQGFSSQLIKWVAALTQAGLSHLNPITYGFKSRMRTLQVLCYAYLMYFSLYSWGMVLHASVPSIGLLSGIKIYPEVYDPWFVVYVIAFISTILENMSESIPEGGSVKTWWVIEYRALMMMGVSAIWLGGVKAIVDKIIGTQGEKLYLSDKAIDKEKLKKYEKGKFDFQGIGILAVPLITFSVLNLVGFLVGINQVLITMKFAGVLGQLLVSSFFVFVVVTVVIDVVSFLKDS>cellulose synthase like_capsicum annum [SEQ ID NO: 42]ATGGAGCTCAACAGATGTACGGTGCAGCAACCTACCACTGCCATATACCGACTACACATGTTTCTCCACTCTCTAATCATGCTTGCATTAGTATACTATCGTTTGTCTAATCTGTTTTACTTCGAAAACGTCCTCACTTTACAAGCATTTGCATGGGGGCTTATCACCTTAGGTGAAATTTGTTTCATTGTCAAGTGGTTCTTTGGTCAAGGGACTCGTTGGCGCCCCGTTGTCAGGGAAGTGTTCCTGGACAATATTACTTGCCAAGATTCCGAGCTGCCCGCACTAGATGTGATGGTTTTCACTGCCAATCCCAAGAAAGAGCCAATTGTGGATGTCATGAACACTGTGATATCCGCAATGGCTCTTGATTACCCGACGGATAAATTGGCTGTGTATCTGGCTGATGATGGAGGATGCCCCTTGACGTTGTACGCCATGGAGGAGGCCTGTTCTTTTGCCAAGTTGTGGCTACCTTTCTGTAGGAAGTATGGAATCAAAACAAGGTGCCCCAAAGCGTTTTTTTCTCCATTAGGAGAAGATGATCGTATCCTTAAGAACGATGACTTTGTAGCTGAAATGAAAGAAATTAAATTAAAATATGAGGAGTTCCAGCAGAATGTAAACCTTGCTGGTGAATCCGGAAAAATCAAAGGTGACGTAGTGCCTGATAGAGCCTCGTTTATTAAGGTAATAAATGACAGGAAAATGGAGAACAAGAAGAGTGCCGACGATATAACGAAAATGCCTTTGCTAGTATACGTATCCCGTGAAAGAAGATTTAACAGTCGTCATCACTTCAAGGGTGGATCTGCAAATGCTCTTCTTCGAGTTTCAGGGATAATGAGTAATGCCCCCTATTTACTGGTCTTAGATTGTGATTTCTTCTGTCATGATCCAACATCAGCTCGGAAGGCAATGTGTTTCCATCTTGATCCAAAACTATCACCTTCCTTAGCTTATGTGCAGTTCCCTCAAGTGTTTTACAATGTCAGCAAGTCCGATATATACGATGTCAAAATTAGACAGGCTTACAAGACAATATGGCACGGAATGGATGGTATCCAAGGCCCAGTGTTATCGGGAACTGGGTATTTTCTGAAGAGGAAAGCGTTATACACGAGTCCAGGTCTAAAGGATGAGTATCTTATTTCACCGGAAAAGCATTTCGGATCAAGTAGAAAGTTCATTGCTTCTCTAGAGGAGAACAATGGTTATGTrAAGCAAGAGAAACTCATAACAGAAGATATTATAGAGGAAGCGAAGACCTTGTCTACTTGTGCATACGAGGATGGTACACGATGGGGCGAAGAGATCGGTTATACCTACAATTGCCATTTGGAGAGCACTTTTACCGGCTATCTTTTGCACTGCAAAGGGTGGACATCAACATATTTGTATCCAGAAAGGCCATCTTTCTTGGGTTGTGCCCCAGTTGATATGCAAGGATTCTCCTCACAACTCACAAAATGGGTTGCTGCACTCACACAAGCTGGTCTATCACATCTCAATCCCATCACTTACGGCATGAAGAGCAGGATTAAGACTATCCAATGCTTGTGCTATGCCTATTTGATGTATTTCTCTCTCTATTCTTGGGGAATGGTTCTGCATGCTAGTGTTCCTTCTATTAGCCTTTTGCTTGGCATTCAAGTCTACCCCGAGGTCTATGATCCATGGTTTGCAGTGTATGTGCTTGCTTTCATATCGACAATTTTGGAGAACATGTCAGAGTCAATTCCAGAAGGCGGTTCAGTTAAAACTTGGTGGATGGAATACAGGGCACTGATGATGATGGGAGTTAGTGCAATATGGTTAGGAGGAGTGAAAGCTATAGTAGAAAAGATCATCGGAACTCAAGGAGAGAAATTATATTTGTCGGACAAAGCAATTGACAAGGAAAAGCTCAAGAAATATGAGAAGGGGAAATTTGATTTCCAAGGGATAGGGATACTTGCTGTTCCATTGATAACATTCTCAGCGTTGAATTTGGTAGGCTTCATGGTTGGAGCTAATCAAGTGATTCTTACTATGAAGTTCGAAGCTTTGCTAGGCCAACTCCTTGTGTCATCCTTCTTCGTCTTTGTGGTGGTCACCGTTGTCATAGATGTCCTATCTTTCTTAAAAGACTCTTAA>cellulose synthase like_capsicum annuum [SEQ ID NO: 43]MELNRCTVQQPTTAIYRLHMFLHSLIMLALVYYRLSNLFYFENVLTLQAFAWGLITLGEICFIVKWFFGQGTRWRPVVREVFLDNITCQDSELPALDVMWTANPKKEPIVDVMNTVISAMALDYPTDKLAVYLADDGGCPLTLYAMEEACSFAKLWLPFCRKYGIKTRCPKAFFSPLGEDDRILKNDDFVAEMKEIKLKYEEFQQNANLAGESGKIKGDVVPDRASFIKYINDRKMENKKSADDITKMPLLVYVSRERRFNSRHHFKGGSANALLRVSGIMSNAPYLLVLDCDFFCHDPTSARKAMCFHLDPKLSPSLAYVQFPQVFYNVSKSDIYDVKIRQAYKTIWHGMDGIQGPVLSGTGYFLKRKALYTSPGLKDEYLISPEKHFGSSRKFIASLEENNGYVKQEKLITEDIIEEAKTLSTCAYEDGTRWGEEIGYTYNCHLESTFTGYLLHCKGWTSTYLYPERPSFLGCAPVDMQGFSSQLTKWVAALTQAGLSHLNPITYGMKSRIKTIQCLCYAYLMYFSLYSWGMVLHASVPSISLLLGIQVYPEVYDPWFAVYVLAFISTILENMSESIPEGGSVKTWVVMEYRALMVIMGVSAIWLGGVKAIVEKIIGTQGEKLYLSDKAIDKEKLKKYEKGKFDFQGIGILAVPLITFSALNLVGFMVGANQVILTMKFEALLGQLLVSSFFVFVVVTVVIDVLSF LKDS

The following sequences were generated for silencing GAME15 in theirrespective plants:

Region used for GAME15 silencing in Tomato [SEQ ID NO: 44]GGCTCTTGATTATCCCACCGATAAATTGGCTGTGTATCTCGCTGATGATGGAGGATGTCCATTGTCGTTGTACGCCATGGAACAAGCGTGTTTGTTTGCAAAGCTATGGTTACCTTTCTGTAGAAACTATGGAATTAAAACGAGATGCCCAAAAGCATTTTTTTCTCCGTTAGGAGATGATGACCGTGTTCTTAAGAATGATGATTTTGCTGCTGAAATGAAAGAAATTAAATTGAAATATGAAGAGTTC CAGCAGAAGGTGGAACATGC Region used for GAME15 silencing in Potato [SEQ ID NO: 45]GGCTCTTGATTATCCTACGGATAAATTGGCTGTGTATCTGGCTGATGATGGAGGATGTCCTTTGTCATTGTACGCCATGGAAGAAGCATGTGTGTTTGCAAAGCTGTGGCTACCTTTCTGTAGGAAGTATGGAATTAAAACTAGATGCCCTAAAGCGTTTTTTTCTCCTTTAGGAGATGATGAACGTGTTCTTAAGAATGATGATTTTGATGCTGAAATGAAAGAAATTAAATTGAAATATGAAGAGTTCCAGCAGAATGTGGAACGTGCTGGTG Region used for GAME15 silencing in Eggplant[SEQ ID NO: 46] GGCTCTTGATTACCCGACCGACAAATTGGCCGTTTATTTGTCTGATGATGGAGGATGCCCCTTGACGTTGTACGCAATGGAGGAAGCTTGTTCCTTTGCCAAGTTGTGGCTACCTTTTTGTAGGAAGTATGGAATCAAAACAAGGTGCCCTAAGGCGTTTTTTTCTCCATTAGGAGAAGATGACCGTGTATTGAAGAGTGATGACTTTGTTTCTGAAATGAAAGAAATGAAGTCAAAATATGAAGAGTTCCAGCAGAACGTGGACCGTGCTGGTGAATCCGGAAAAATCAAAGGTGACGTAGTGCCTGATAGACCCGCGTTTCTTAAGGTACTAAATGACAGGAAGACGGAGAACGAGAAGAGTGCAGACGATTTAACTAAAATGCCTTTGCTAGTATACGTATCCCGTGAAAGAAGAACTCACCGTCGCCATCACTTCAAGGGTGG 

RNAi lines for the GAME15 gene in tomato and potato were generated.GAME15-RNAi transgenic tomato plants showed severe reduction inα-tomatine and downstream SGAs in leaves; α-tomatine was not detected inGAME15-silenced green fruit. Furthermore, no esculeosides or other SGAswere detected during tomato fruit developmental stages (e.g., breakerand red fruit). In addition, a 15-20 fold increase in cholesterol, whichis a precursor for SGAs was observed in leaves and green fruit ofGAME15-RNAi tomato plants. In potato, silencing of GAME15 resulted in amajor reduction in α-chaconine and α-solanine, while the cholesterolpool in these lines increased.

Example 6 Generation of GAME15-RNAi Transgenic Tomato Potato andEggplant Plants

The GAME15-RNAi construct was generated by introducing a selectedfragment (silencing sequences SEQ ID NO: 44 (tomato), SEQ ID NO: 45(potato), and SEQ ID NO: 46 (eggplant)) to pENTR/D-TOPO (invitrogen) (byNotI and AscI) and further subcloning of this fragment to the pK7GWIWG2(II) binary vector using the Gateway LR Clonase II enzyme mix(invitrogen). The vector was transformed into tomato, potato andeggplant as described previously (Itkin et al. 2011. The Plant Cell23:4507-25; Sonawane et al. 2018. PNAS 115(23): E5418-E5428). PositiveGAME25-downregulated lines were further used for LC-MS analysis.

Example 7 GAME15-Silenced Tomato Plants Showed Severely Reduced SGAProfile

In order to determine the precise role of GAME15 in SGA metabolism,GAME15-RNAi (GAME15i) transgenic tomato lines (#21, #22 and #23) weregenerated using the tomato silencing sequence above (SEQ ID NO: 44).

GAME15-RNAi leaves showed severe reduction in α-tomatine, compared withwild-type tomato leaves (FIG. 15A). Furthermore, the SGAs profile ofGAME15i fruit was subsequently compared to wild-type ones at differentstages of development and ripening. During the transition from green tored fruit in tomato, α-tomatine is converted to esculeosides andlycoperosides, while dehydrotomatine is converted to dehydroesculeosidesand dehydrolycoperosides (FIG. 14A).

GAME15i green and red fruits did not show any trace of SGAs (e.g.,α-tomatine or Esculeoside A) suggesting complete loss of SGAs in tomatofruits due to GAME15i silencing (FIGS. 15B and 15C).

Example 8 Altering GAME15 Expression has Major Impact on SGAs in Potato

Similar to tomato, GAME15i was also silenced in potato (#1, #2, and #3)to determine its effect on potato SGAs metabolism, using the potatosilencing sequence above (SEQ ID NO: 45).

Silencing of GAME15 in potato resulted in drastic reduction inα-chaconine (shaded bars) and α-solanine (open bars), major SGAs inpotato leaf tissue (FIG. 16), in comparison with potato leaf tissue ofthe wild-type.

Example 9 High Cholesterol Accumulation in GAME15-Silenced Tomato Leaves

Cholesterol serves as a key precursor in the biosynthesis of SGAs(Sonawane et al., 2016, Nat. Plants 3: 16205). As severe reduction andsubsequent complete loss of SGAs was observed in GAME15i-silenced tomatoplants, the cholesterol levels in these plants were examined. An˜15-20-fold increase in cholesterol (SGA precursor) was observed inleaves of GAME15i-silenced tomato plants compared to the leaves ofwild-type tomato plants (FIG. 17).

Example 10 Altering GAME15 Expression and Observing its Impact on SGAsin Eggplant

Similar to potato, GAME15i is also silenced in eggplant to determine itseffect on potato SGAs metabolism, using the eggplant silencing sequenceabove (SEQ ID NO: 46).

The effect of silencing of GAME15 in eggplant is observed with respectto reduced levels of α-solasonine and/or α-solamargine in comparisonwith wild-type eggplant (FIG. 14C).

Example 11 Overexpression of GAME15 in Tomato, Potato, and Eggplant

Alternatively, tomato, potato, and/or eggplant plants are geneticallymodified or gene edited to overexpress GAME15.

To increase production of α-tomatine and esculeosides and/orlycoperosides in tomato plants (FIG. 14A), tomato plants are geneticallymodified or gene edited to overexpress GAME15.

To increase production of α-solanine and/or α-chaconine in potato plants(FIG. 14B), potato plants are genetically modified or gene edited tooverexpress GAME15.

To increase production of α-solasonine and/or α-solamargine in eggplant(FIG. 14C), eggplant plants are genetically modified, or gene edited tooverexpress GAME15.

Example 12 Plants and Crops with Modified Levels and Compositions ofSGAs

Based on the foregoing, Solanaceous plants (e.g., tomato, potato,eggplant, and/or pepper plants) and/or crops are prepared, such asthrough classical breeding or genetic engineering (e.g., geneticallymodified or transgenic plants, gene edited plants, and the like), withmodified levels and compositions of SGAs, conferring on the plant achemical barrier against a broad range of insects and other pathogensand/or removing anti-nutritional compounds (e.g., chaconine and/orsolanine from potato).

Furthermore, high cholesterol or high phytosterol tomato lines are usedto engineer high value steroidal compounds (e.g., pro-vitamin and/ordiosgenin), such as through synthetic biology tools.

In addition, high phytosterol (e.g., phytocholesterol) lines are used toproduce components used in cosmetic products.

In other instances, Solanaceous plants (e.g., tomato, potato, eggplant,and/or pepper plants) and/or crops are prepared with increased levels ofSGAs and/or decreased levels of phytosterols.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1. A genetically modified plant comprising at least one cell havingaltered expression of at least one gene selected from the groupconsisting of a gene encoding at least one cellulose synthase-likeprotein compared to its expression in a corresponding unmodified plant,wherein the genetically modified plant has an altered content of atleast one steroidal alkaloid or a glycosylated derivative thereof, or ofat least one steroidal saponin or a glycosylated derivative thereof,compared to the corresponding unmodified plant.
 2. The geneticallymodified plant of claim 1, wherein the cellulose synthase-like proteinis a GAME15 protein, wherein: (a) the amino acid sequence of thecellulose synthase-like protein of the corresponding unmodified plantcomprises the sequence set for cellulose synthase-like protein is atleast 80% homologous to the amino acid sequence set forth in any one ofSEQ ID NOS: 33, 35, 37, 39, 42, or 43; or (b) wherein the polynucleotideencoding the cellulose synthase-like protein of the correspondingunmodified plant comprises the nucleic acid sequence set forth in anyone of SEQ ID NOS: 32, 34, 36, 38, 40, or
 42. 3. The geneticallymodified plant of claim 1, wherein the expression of the at least onegene or any combination thereof is altered, the altering comprisingmutagenizing the at least one gene, wherein the mutagenesis comprisesintroduction of one or more point mutations, or genome editing, or useof a bacterial CRISPR/CAS system, or a combination thereof.
 4. Thegenetically modified plant of claim 1, wherein expression of the geneencoding the at least one cellulose synthase-like protein is reducedcompared to its expression in the corresponding unmodified plant,thereby the genetically modified plant comprises reduced content atleast one steroidal alkaloid or a glycosylated derivative thereof, or ofat least one steroidal saponin or a glycosylated derivative thereof,compared to the corresponding unmodified plant.
 5. The geneticallymodified plant of claim 4, wherein the genetically modified plant is atransgenic plant comprising at least one cell comprising at least onesilencing molecule targeted to a polynucleotide encoding at least onecellulose synthase-like protein, or wherein the transgenic plantcomprises a polynucleotide encoding a cellulose synthase-like protein,wherein expression of the polynucleotide is selectively silenced,repressed, or reduced.
 6. The genetically modified plant of claim 5,wherein the transgenic plant comprises a polynucleotide encoding acellulose synthase-like protein, wherein the polynucleotide has beenselectively edited by deletion, insertion, or modification to silence,repress, or reduce expression thereof, or wherein the geneticallymodified plant is a progeny of the gene edited plant.
 7. The geneticallymodified plant of claim 5, wherein the transgenic plant comprises atleast one cell comprising at least one silencing molecule targeted to aGAME15 gene.
 8. The genetically modified plant of claim 5, wherein: (a)wherein the silencing molecule comprises a polynucleotide having anucleic acid sequence substantially complementary to a region of theGAME15 gene having the nucleic acid sequence set forth in any one SEQ IDNOS: 32, 34, 36, 38, 40, or 42 or a complementary sequence thereof; or(b) the silencing molecule is selected from the group consisting of anRNA interference molecule and an antisense molecule, or wherein thesilencing molecule is a component of a viral induced gene silencingsystem.
 9. The genetically modified plant of claim 8, wherein: (a) thesilencing molecule is targeted to a GAME15 fragment having the nucleicacid sequence set forth in SEQ ID NO: 44 or a complementary sequencethereof; (b) the silencing molecule is targeted to a GAME15 fragmenthaving the nucleic acid sequence set forth in SEQ ID NO: 45 or acomplementary sequence thereof; or (c) the silencing molecule istargeted to a GAME15 fragment having the nucleic acid sequence set forthin SEQ ID NO: 46 or a complementary sequence thereof.
 10. Thegenetically modified plant of claim 5, wherein the genetically modifiedplant is a Solanaceae plant having a reduced content of at least onesteroidal glycoalkaloid selected from the group consisting ofα-tomatine, tomatidine, α-chaconine, α-solanine, α-solasonine,α-solmargine, and derivatives thereof, compared to a correspondingunmodified plant.
 11. The genetically modified plant of claim 5, whereinthe genetically modified plant further comprises an elevated content ofa phytosterol or a derivative thereof, a cholesterol or a derivativethereof, a phytocholesterol or a derivative thereof, a cholestenol or aderivative thereof, a phytocholestanol or a derivative thereof, or asteroidal saponin or a derivative thereof compared to a correspondingunmodified plant.
 12. The genetically modified plant of claim 10,wherein the plant is a Solanaceae plant selected from the groupconsisting of tomato, potato, eggplant, and pepper.
 13. The geneticallymodified plant of claim 12, wherein the plant is a Solanaceae plant: (a)the plant is a tomato plant comprising a reduced content of α-tomatine,tomatidine, or derivatives thereof or an elevated content of aphytosterol, a phytocholesterol or cholesterol, a phytocholestenol orcholestenol, a steroidal saponin, or derivative thereof; (b) the plantis a potato plant comprising a reduced content of α-chaconine,α-solanine, or derivatives thereof; or (c) the plant is an eggplantplant comprising a reduced content of α-solasonine, α-solamargine, orderivatives thereof.
 14. The genetically modified plant of claim 1,wherein expression of the gene encoding the at least one cellulosesynthase-like protein is elevated compared to its expression in thecorresponding unmodified plant, thereby the genetically modified plantcomprises elevated content at least one steroidal alkaloid or aglycosylated derivative thereof, or of at least one steroidal saponin ora glycosylated derivative thereof, compared to the correspondingunmodified plant.
 15. The genetically modified plant of claim 14,wherein the transgenic plant comprises a polynucleotide encoding acellulose synthase-like protein, wherein expression of thepolynucleotide is selectively increased.
 16. The genetically modifiedplant of claim 15, wherein the transgenic plant comprising at least onecell comprising at least one transcribable polynucleotide encoding atleast one protein selected from the group consisting of at least one acellulose synthase-like protein, wherein: (a) the cellulosesynthase-like protein is a GAME15 protein; or (b) the transcribablepolynucleotide comprises the nucleic acid sequence set forth in any oneof SEQ ID NOS: 32, 34, 36, 38, 40, or
 42. 17. The genetically modifiedplant of claim 14, wherein the genetically modified plant is aSolanaceae plant having an elevated content of at least one steroidalglycoalkaloid selected from the group consisting of α-tomatine,tomatidine, α-chaconine, α-solanine, α-solasonine, α-solmargine, andderivatives thereof, compared to a corresponding unmodified plant. 18.The genetically modified plant of claim 14, wherein the geneticallymodified plant further comprises a reduced content of a phytosterol or aderivative thereof, a cholesterol or a derivative thereof, aphytocholesterol or a derivative thereof, a cholestenol or a derivativethereof, a phytocholestanol or a derivative thereof, or a steroidalsaponin or a derivative thereof compared to a corresponding unmodifiedplant.
 19. The genetically modified plant of claim 17, wherein the plantis a Solanaceae plant selected from the group consisting of tomato,potato, eggplant, and pepper.
 20. A method of reducing the content of atleast one steroidal alkaloid or a glycosylated derivative thereof, or ofat least one steroidal saponin or a glycosylated derivative thereof, ina modified plant, the method comprising (a) transforming at least oneplant cell with at least one silencing molecule targeted to a nucleicacid sequence encoding at least one protein comprising a cellulosesynthase-like protein; or (b) mutagenizing at least one gene or acombination of genes, the genes encoding at least one protein selectedfrom the group consisting of cellulose synthase-like proteins, whereinthe mutagenesis comprises introduction of one or more point mutationsinto the gene, or genome editing, or use of a bacterial CRISPR/CASsystem, or a combination thereof, wherein expression of the geneencoding the at least one cellulose synthase-like protein is reduced inthe modified plant compared to its expression in a correspondingunmodified plant, thereby the modified plant comprises reduced contentat least one steroidal alkaloid or a glycosylated derivative thereof, orof at least one steroidal saponin or a glycosylated derivative thereof,compared to the corresponding unmodified plant.
 21. The method of claim20, wherein the cellulose synthase-like protein is a GAME15 protein,wherein: (a) the amino acid sequence of the cellulose synthase-likeprotein of the corresponding unmodified plant comprises the sequence setfor cellulose synthase-like protein is at least 80% homologous to theamino acid sequence set forth in any one of SEQ ID NOS: 33, 35, 37, 39,42, or 43; or (b) the polynucleotide encoding the cellulosesynthase-like protein of the corresponding unmodified plant comprisesthe nucleic acid sequence set forth in any one of SEQ ID NOS: 32, 34,36, 38, 40, or
 42. 22. The method of claim 20, wherein: (a) thesilencing molecule is targeted to a GAME15 fragment having the nucleicacid sequence set forth in SEQ ID NO: 44 or a complementary sequencethereof; (b) the silencing molecule is targeted to a GAME15 fragmenthaving the nucleic acid sequence set forth in SEQ ID NO: 45 or acomplementary sequence thereof; or (c) the silencing molecule istargeted to a GAME15 fragment having the nucleic acid sequence set forthin SEQ ID NO: 46 or a complementary sequence thereof.
 23. The method ofclaim 20, wherein the modified plant is a Solanaceae plant having areduced content of at least one steroidal glycoalkaloid selected fromthe group consisting of α-tomatine, tomatidine, α-chaconine, α-solanine,α-solasonine, α-solmargine, and derivatives thereof, compared to thecorresponding unmodified plant.
 24. The method of claim 20, wherein themodified plant further comprises an elevated content of a phytosterol ora derivative thereof, a cholesterol or a derivative thereof, aphytocholesterol or a derivative thereof, a cholestenol or a derivativethereof, a phytocholestanol or a derivative thereof, or a steroidalsaponin or a derivative thereof compared to a corresponding unmodifiedplant.
 25. The method of claim 23, wherein the modified plant is aSolanaceae plant selected from the group consisting of tomato, potato,eggplant, and pepper.
 26. The method of claim 23, wherein: (a) the plantis a tomato plant comprising a reduced content of α-tomatine,tomatidine, or derivatives thereof or an elevated content of aphytosterol, a phytocholesterol or cholesterol, a phytocholestenol orcholestenol, a steroidal saponin, or derivative thereof; (b) the plantis a potato plant comprising a reduced content of α-chaconine,α-solanine, or derivatives thereof; or (c) the plant is an eggplantplant comprising a reduced content of α-solasonine, α-solamargine, orderivatives thereof.
 27. A method of producing at least one phytosterolin a modified plant, the method comprising (a) transforming at least oneplant cell with at least one silencing molecule targeted to a nucleicacid sequence encoding at least one protein comprising a cellulosesynthase-like factor; or (b) mutagenizing at least one gene or acombination of genes, the genes encoding at least one protein selectedfrom the group consisting of cellulose synthase-like factors, whereinthe mutagenesis comprises introduction of one or more point mutationsinto the gene, or genome editing, or use of a bacterial CRISPR/CASsystem, or a combination thereof, wherein expression of the geneencoding the at least one cellulose synthase-like protein is reduced inthe modified plant compared to its expression in a correspondingunmodified plant, thereby the modified plant comprises reduced contentat least one steroidal alkaloid or a glycosylated derivative thereof, orof at least one steroidal saponin or a glycosylated derivative thereof,compared to the corresponding unmodified plant; and wherein the modifiedplant further comprises an elevated content of a phytosterol or aderivative thereof, a cholesterol or a derivative thereof, aphytocholesterol or a derivative thereof, a cholestenol or a derivativethereof, a phytocholestanol or a derivative thereof, or a steroidalsaponin or a derivative thereof compared to a corresponding unmodifiedplant.
 28. The method of claim 27, wherein the cellulose synthase-likeprotein is a GAME15 protein wherein: (a) the amino acid sequence of thecellulose synthase-like protein of the corresponding unmodified plantcomprises the sequence set for cellulose synthase-like protein is atleast 80% homologous to the amino acid sequence set forth in any one ofSEQ ID NOS: 33, 35, 37, 39, 42, or 43; or (b) wherein the polynucleotideencoding the cellulose synthase-like protein of the correspondingunmodified plant comprises the nucleic acid sequence set forth in anyone of SEQ ID NOS: 32, 34, 36, 38, 40, or
 42. 29. The method of claim27, further comprising purifying the phytosterol extracted from thetransformed plant.
 30. The method of claim 27, wherein the phytosterolcomprises phytocholesterol.
 31. The method of claim 27, wherein: (a) thesilencing molecule is targeted to a GAME15 fragment having the nucleicacid sequence set forth in SEQ ID NO: 44 or a complementary sequencethereof; (b) the silencing molecule is targeted to a GAME15 fragmenthaving the nucleic acid sequence set forth in SEQ ID NO: 45 or acomplementary sequence thereof; or (c) the silencing molecule istargeted to a GAME15 fragment having the nucleic acid sequence set forthin SEQ ID NO: 46 or a complementary sequence thereof.
 32. The method ofclaim 27, wherein the modified plant is a Solanaceae plant.
 33. Themethod of claim 32, wherein the Solanaceae plant is selected from thegroup consisting of tomato, potato, eggplant, and pepper.